NOVEL SECRETED ANTIGENS FOR DIAGNOSIS OF ACTIVE BABESIA MICROTI AND BABESIA DUNCANI INFECTION IN HUMANS AND ANIMALs
20220236268 · 2022-07-28
Inventors
Cpc classification
International classification
Abstract
In various aspects and embodiments, the invention provides a method of detecting a Babesia infection in a subject comprising detecting one or more peptides selected from SEQ ID NOs: 1-62 in a biological sample collected from the subject; wherein detecting one or more of the peptides indicates the presence of Babesia infection.
Claims
1. A method of detecting a Babesia infection in a subject, the method comprising: detecting whether one or more secreted antigens selected from SEQ ID NOs: 1-62 are present in a biological sample collected from the subject; wherein detecting presence of one or more of the antigens in the biological sample indicates that the subject has a Babesia infection.
2. The method of claim 1, wherein the Babesia infection comprises Babesia microti or Babesia duncani.
3. The method of claim 1, wherein the biological sample comprises one or more selected from the group consisting of: a blood sample, an erythrocyte sample, a leukocyte sample, a plasma sample, a urine sample, and a saliva sample.
4. The method of claim 1, wherein the one or more antigens further comprises BmGPI12.
5. The method of claim 1, wherein the subject is a human.
6. The method of claim 1, wherein the subject is a mammal known to carry a Babesia parasite.
7. The method of claim 1, wherein the one or more antigens are detected by one or more antibody-based techniques selected from the group consisting of: Western blot, immunofluorescent assay, IEM, ELISA, PCR amplification-based immunoassay and immunoprecipitation.
8. A diagnostic tool for identifying or diagnosing a babesiosis infection, the tool comprising: an assay platform, and an immunologic agent having specificity for one or more Babesia antigens selected from SEQ ID NOs: 1-62.
9. The diagnostic tool of claim 8, wherein the babesiosis infection comprises a Babesia microti infection or a Babesia duncani infection.
10. The diagnostic tool of claim 8, wherein the assay platform comprises one or more selected from the group consisting of: an enzyme-based assay, a radioimmunoassay, a PCR amplification-based immunoassay, a fluorogenic immunoassay, a chemiluminescence-based assay, and immunoblotting assay.
11. The diagnostic tool of claim 8, wherein the immunologic agent comprises one or more of antibodies or antibody fragments.
12. A method of treating, ameliorating, and/or preventing a Babesia infection in a subject in need thereof, the method comprising: obtaining a first sample from a subject at a first time point; assaying the sample using the diagnostic tool of claim 8 to detect the presence or absence of an infection relative to a comparator control, administering one or more therapeutic agents to the subject; obtaining a second sample from the subject at a second time point, wherein the second time point comprises one or more time points after the first time point; and assaying the second sample obtained from the subject at the second time point using the diagnostic tool of claim 8 to detect the presence or absence of an infection relative to a comparator control.
13. The method of claim 12, wherein the sample comprises a blood sample.
14. The method of claim 12, wherein the infection comprises a Babesia microti infection or a Babesia duncani infection.
15. The method of claim 12, wherein the first time point is before administration of the therapeutic agent.
16. The method of claim 13, wherein the second time point is after administration of the therapeutic agent.
17. A method of treating, ameliorating, or preventing a Babesia infection in a subject, the method comprising: detecting presence of one or more peptides selected from SEQ ID NOs: 1-62 in a biological sample collected from the subject; and administering to the subject at least one anti-protozoan therapeutic agent.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
Definitions
[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.
[0021] As used herein, each of the following terms has the meaning associated with it in this section.
[0022] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
[0023] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
[0024] As used herein, to “alleviate” a disease, defect, disorder or condition means reducing the severity of one or more symptoms of the disease, defect, disorder or condition.
[0025] The term “antigen” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen.
[0026] Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.
[0027] The term “epitope” as used herein is defined as a small chemical molecule on an antigen that can elicit an immune response, inducing B and/or T cell responses. An antigen can have one or more epitopes. Most antigens have many epitopes; i.e., they are multivalent. In general, an epitope is roughly about 10 amino acids and/or sugars in size. Preferably, the epitope is about 4-18 amino acids, more preferably about 5-16 amino acids, and even more preferably 6-14 amino acids, more preferably about 7-12, and most preferably about 8-10 amino acids. One skilled in the art understands that generally the overall three-dimensional structure, rather than the specific linear sequence of the molecule, is the main criterion of antigenic specificity and therefore distinguishes one epitope from another. Based on the present disclosure, a peptide used in the present invention can be an epitope.
[0028] The term “immune response” as used herein is defined as a cellular response to an antigen that occurs when lymphocytes identify antigenic molecules as foreign and induce the formation of antibodies and/or activate lymphocytes to remove the antigen.
[0029] By the term “modified” as used herein, is meant a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids.
[0030] By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and/or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and/or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.
[0031] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
[0032] By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.
[0033] As used herein, the terms “subject” and “patient” are used interchangeably. As used herein, a subject is preferably a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) and a primate (e.g., monkey and human), most preferably a human.
[0034] As used herein, to “treat” means reducing the frequency with which symptoms of a disease, defect, disorder, or adverse condition, and the like, are experienced by a patient.
[0035] The term “therapeutic” as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.
[0036] As used herein, a “therapeutically effective amount” is the amount of a composition of the invention sufficient to provide a beneficial effect to the individual to whom the composition is administered.
[0037] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
DESCRIPTION
[0038] The present invention relates to compositions and methods for detecting, diagnosing, screening for, treating and/or reducing Babesia infections such as babesiosis in a subject. The Babesia infection may include Babesia microti (B. microti) infections, Babesia duncani (B. duncani) infections, and/or combinations thereof. The subject may include a mammal. In certain embodiments, the subject is a human.
Compositions and Methods for Detecting and/or Diagnosing an Infection
[0039] In certain embodiments, the present invention provides compositions and methods for detecting an infection in a subject. The infection includes parasitic infections such as Babesia infections caused by one or more Babesia strains. The Babesia infection may include Babesia microti (B. microti) infections, Babesia duncani (B. duncani) infections, and/or combinations thereof.
[0040] In certain embodiments, the compositions includes one or more compositions for detecting one or more secreted antigens. The one or more antigens include one or more antigens generated by one or more cells infected with a Babesia infection (e.g. B. duncani infection, B. microti infection and/or combination thereof). The one or more antigens may include one or more peptides including one or more of SEQ ID NOs: 1-62.
Compositions for Detecting/Diagnosing a Babesia Infection
[0041] In certain aspects, the present invention provides compositions for detecting and/or diagnosing and infection in a subject. The compositions may include one or more immunologic agents for detecting one or more antigens or peptides generated by one or more cells. The one or more antigens or peptides may be detected in a sample obtained from a subject with an infection.
[0042] In various embodiments, the one or more immunologic agents include one or more immunologic agents that have specificity for one or more antigens generated by the one or more cells. For example the one or more immunologic agents may include one or more of an antibody, an antibody fragment, antisense peptide, synthetic peptide, synthetic antibody, synthetic antibody fragment, synthetic hybridized antibody or antibody fragment. The one or more immunologic agents detect, bind to, or interact with one or more peptides or antigens generated by one or more cells infected with one or more of B. duncani, B. microti, or both. The one or more peptides of antigens may include one or more selected from SEQ. ID. NOs: 1-46, 47-62. In some embodiments, one or more antigens or peptides selected from SEQ. ID. NOs: 1-46 may be used to detect a B. duncani infection in a sample obtained from a subject. In some embodiments, one or more antigens or peptides selected from SEQ. ID. NOs: 47-62 may be used to detect a B. microti infection in a sample obtained from a subject.
[0043] In certain embodiments, the biological sample includes one or more of blood, urine, saliva, feces, lymph, bile and the like, and/or one or more combinations thereof. The sample may include one or more of a whole blood sample, a plasma sample, a serum sample, a hemolysate sample, and the like. In some embodiments, the one or more infected cells include one or more blood-derived cells such as one or more of erythrocyte, leukocyte, plasma cell, platelets, and the like.
[0044] Methods for Detecting/Diagnosing a Babesia Infection
[0045] In certain embodiments, the present invention relates to methods for detecting and/or diagnosing one or more Babesia infections including babesiosis, B. duncani infection, B. microti infection, and/or combinations thereof.
[0046] In certain embodiments, the methods include obtaining one or more biological samples obtained from a subject. The one or more biological samples may include one or more of blood, urine, saliva, feces, lymph, bile and the like, and/or one or more combinations thereof. The blood sample may include whole blood sample, a plasma sample, a serum sample, a hemolysate sample, and the like. The subject may include a human subject. The subject may have a known or suspected infection of one or more Babesia strains including one or more of B. duncani, B. microti, and/or combinations thereof.
[0047] Embodiments of the methods include detecting one or more infections in one or more biological samples obtained from a subject. The one or more infections include one or more of babesiosis, B. duncani infection, B. microti infection, and/or combinations thereof. The infection may be detected using one or more immunologic agent having specificity for one or more antigens or peptides including one or more of SEQ. ID. NOs: 1-46, 47-62, and/or one or more combinations thereof. In certain embodiments, the one or more antigens or peptides are detected using one or more assay platforms including for example, an enzyme-based assay, a radioimmunoassay, a PCR amplification-based assay, a fluorogenic immunoassay, a chemiluminescence-based assay, immunoblotting assay, and combinations thereof. The assay may include one or more of Western blot, immunofluorescence, immune-electron microscopy, ELISA, immunoprecipitation, and the like.
[0048] In certain embodiments, the one or more antigens or peptides are detected relative to a comparator control. In certain embodiments, an infection is detected if the signal is greater than or less than a threshold value, a difference relative to a comparator control, or the like.
[0049] Embodiments of the methods include measuring one or more biological samples obtained from a subject in order to detect the presence of an infection. Embodiments of the methods include measuring one or more biological samples obtained from a subject in order to evaluate the efficacy of one or more therapeutic agents. In various embodiments, the sample is a blood sample, a sera sample, and/or one or more samples containing one or more other suitable bodily fluids. In various embodiments, the subject is a subject that is healthy, a subject that is confirmed to be infected, a subject that is suspected to be infected, a subject that has been treated for an infection and is believed to no longer be infected, and the like. The subject may be evaluated by assaying the one or more samples obtained from the subject. The one or more samples may be evaluated at one or more time points in order to evaluate the status of an infection and/or the efficacy of one or more treatments for the infection. In various embodiments, the one or more time points include prior to an intentional infection, such as about 24 hours prior to infection, from about 24 hours to about 12 hours prior to infection, from about 12 hours to about 8 hours prior to infection, from about 8 hours to about 4 hours prior to infection, from about 4 hours to about 1 hour prior to infection, less than about 1 hour prior to infection, and any and all intervals and increments therebetween. The one or more time points include at the time of infection, up to 1 hour post-infection, from about 1 hour to about 1 day post-infection, from about 1 day to about 2 days post infection, about 2 days to about 3 days post-infection, from about 3 days to about 4 days post-infection, from about 4 days to about 5 day post-infection, from about 5 days to about 6 days post-infection, from about 6 day to about 7 days post-infection, from about 1 week to about 2 weeks post-infection, from about 2 weeks to about 4 weeks post-infection, from about 4 weeks to about 6 weeks post-infection, and so on. In various embodiments, the one or more time points include at the time of infection, immediately prior to administering a therapeutic agents, at the time of administering a therapeutic agent, and at one or more time points after administration of a therapeutic agent. The one or more time points may include up to 24 hours prior to administration of a therapeutic agent, from about 24 hours to about 12 hours prior to administration of a therapeutic agent, from about 12 hours to about 8 hours prior to administration of a therapeutic agent, from about 8 hours to about 4 hours prior to administration of a therapeutic agent, from about 4 hours to about 1 hour prior to administration of a therapeutic agent, less than about 1 hour prior to administration of a therapeutic agent, and any and all increments and intervals therebetween. In various embodiments, the one or more time points include: up to 1 hour post-administration, from about 1 hour to about 12 hours post-administration, from about 12 hours to about 24 hours post-administration, from about 1 day to about 2 days post-administration, from about 2 day to about 5 days post-administration, from about 5 days to about 7 days post-administration, from about 7 days to about 14 days post-administration, from about 14 day to about 28 days post-administration, from about 28 days to about 42 days post-administration, and so on. The one or more time points may include from about 1 week to about 2 weeks post-administration, from about 2 weeks to about 4 weeks post-administration, from about 4 weeks to about 6 weeks post-administration, and so on.
[0050] The methods include detecting an infection in a subject using one or more techniques as described herein. The methods include administering to the subject an effective amount of at least one therapeutic agent including one or more anti-protazoan therapeutic agents. In various embodiments, the anti-protazoan therapeutic agent includes one or more anti-protazoan agents as understood in the art.
EXPERIMENTAL EXAMPLES
[0051] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
Example 1
[0052] The apicomplexan parasite Babesia microti is the primary agent of human babesiosis, a malaria-like illness and potentially fatal tick-borne disease. Unlike its close relatives, the agents of human malaria, B. microti develops within human and mouse red blood cells in the absence of a parasitophorous vacuole, and its secreted antigens lack trafficking motifs found in malarial secreted antigens. Here, it is shows that after invasion of erythrocytes, B. microti undergoes a major morphogenic change during which it produces an interlacement of vesicles (IOV); the IOV system extends from the plasma membrane of the parasite into the cytoplasm of the host erythrocyte.
Methods
[0053] Parasite Strains
[0054] B. microti isolates used in this study are LabS1 and PRA99. These strains were maintained in rag2−/− Knockout (B6.12956-Rag2tmlFwa N12) and SCID (severe combined immunodeficiency) C.B17 SCID C.B-Igh-1b/IcrTac-Prkdcscid and CB17/Icr-Prkdcscid/IcrIcoCrl mice as previously described. Parasitemia was determined by thin blood recombinant protein consisting of amino acids 1-302 of BmGPI12, BmIPA48 (Genebank ID # XP_021338473; EupathDB ID #: BMR1_03g00947) peptide CNKIKTDGGKVDSNS, BmRON2 peptide NKIKTDGGKVDSNS. Monoclonal anti-mouse TER-119 (INVITROGEN®) was used as a control.
[0055] Plasma Collection and Fractionation Studies
[0056] Blood from uninfected or B. microti-infected animals was collected by cardiac puncture and stored in tubes containing K.sub.2EDTA (dipotassium ethylenediaminetetraacetate) solution. For plasma separation, samples were spun down at 1,300 rpm (200×g) for 20 minutes at room temperature. Plasma or supernatant was removed and added to new 1.7 ml microcentrifuge tubes. The remaining cell pellet was washed twice with PBS supplemented with 1% saponin, incubated on ice for 30 minutes and spun at 9,300×g for 10 minutes at 4° C. The resulting supernatant (hemolysate) was collected and the remaining pellet (uninfected) or parasite (infected) fractions were washed twice with PBS and spun at 9,300×g for 10 minutes at 4° C. Plasma (S), hemolysate (H) and pellet (P) fractions were mixed with Lammeli buffer, separated on SDS-PAGE and analyzed by immunoblotting.
[0057] Isolation of Vesicles from Plasma
[0058] IOVs were isolated from the plasma of uninfected mice or mice infected with B. microti by sequential centrifugations following the protocol for the isolation of exosomes with some modifications. Briefly, 400 μm plasma from animals was diluted with 5 ml PBS and centrifuged at 500×g for 30 minutes and then at 16,000×g for 45 min to remove microvesicles. IOVs were pelleted with ultracentrifugation (UC) at 120,000×g for 14 hours at 4° C. using a SORVALL™ MTX 150 Micro-Ultracentrifuge with a S52-ST Swinging-Bucket Rotor (THERMO FISHER SCIENTIFIC). The resulting pellet (P1) was collected and the supernatant was spun again using the same conditions. The resulting pellet (P2) and supernatant (Us) fractions were collected.
[0059] Immunoblot Analysis
[0060] Equal concentrations of plasma (prior to ultracentrifugation), supernatant (Us) and pellet (Up) fractions obtained after UC were analyzed by immunoblotting. To obtain equal concentrations, supernatant (Us) fractions were concentrated with 20% trichloroacetic acid. The pellet fractions (P1 and P2) were diluted further and combined as a single pellet (Up). All the samples were resuspended in Lammeli-buffer and loaded on 10% Mini-PROTEAN® TGX (BIO-RAD LABORATORIES®, Hercules, Calif.) and transferred to nitrocellulose membranes. Membranes were blocked in 5% milk and incubated with a rabbit anti-BmGPI12 serum (1:250) or pre-immune serum (1:250 dilution) overnight at 4° C. The next day, membranes were washed in TBS-T and incubated with ECL-horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:10,000 dilution) for 1 hour. Following additional washes, the membranes were incubated with ECL western blotting detection reagents (GE HEALTHCARE®, Amersham, UK) and exposed to X-ray film using KODAK® autoradiography. The same method was used for immunoblotting of parasite and erythrocyte membrane (P), erythrocyte cytoplasm or hemolysate (H) and plasma or supernatant (S) fractions. Polyclonal antibodies including rabbit anti-BmIPA48 (1:100) and rabbit anti-BmRON2 (1:100) and their corresponding pre-immune sera and monoclonal anti-mouse TER-119 antibody (1:500) were also analyzed by immunoblotting.
[0061] Immunofluorescence Assay (IFA)
[0062] Rabbit anti-BmGPI12 (BmSA-1) antibodies were used to assess the localization of BmGPI12 in B. microti LabS1 and PRA99. Rabbit anti-BmIPA48 serum was used to evaluate the localization of BmIPA48 in LabS1. Blood smears were prepared from retro orbital bleeding (after centrifugation at 1,500 rpm for 2 min (MINISPIN PLUS®, EPPENDORF™) on thin 22 mm×22 mm microscopy cover glasses (12-542-B; FISHERBRAND®) and immediately transferred into a 6-well plate (COSTAR®, CORNING®, Inc.) which was put on a cold metal plate on ice. Afterwards 6-well plates with the blood smears were stored at 4° C. before being further processed. To perform the IFA, thin blood smears were removed from the 4° C. storage and fixed in 1% formaldehyde (28908; THERMO SCIENTIFIC®) diluted in PBS (10010-023; GIBCO®) at 37° C. (PRECISION® mechanical convection incubator, model 6 LM) for 30 minutes followed by three brief rinses in PBS. Smears were then incubated for 1 hour in a blocking buffer (5% heat-inactivated fetal bovine serum (16000-044, GIBCO®) 5% normal goat serum (16210-072; GIBCO-BRL®) and 0.1% saponin (10% stock solution in PBS) (57900-100G; SIGMA®)) at 37° C. and then once rinsed in wash buffer (0.5% fetal bovine serum, 0.5% normal goat serum, 0.05% saponin). To target the mouse erythrocyte membrane, FITC anti-mouse TER-119/Erythroid Cells; Clone: TER-119 (116206; BIOLEGEND®) was used at a 1:1,000 dilution. The anti-BmGPI12 or anti-BmIPA48 antibodies were used at 1:1,000 dilution.
[0063] The smears were either incubated overnight at 4° C. while being rocked gently (Orbi-Blotter; BENCHMARK®) or for 1 hour at 37° C. The next day, slides were washed three times for 2 minutes each with wash buffer and incubated with secondary antibody, Goat anti-rabbit IgG (H+L) rhodamine conjugate (31670, Invitrogen) (1:1,000) at 37° C. for 1 hour and washed three times for 2 minutes each in wash buffer. After three brief rinses in PBS and one brief rinse in ddH.sub.2O, the coverslips were mounted on sandblasted single frosted pre-cleaned microscope slides (421-004T; THERMO SCIENTIFIC®) using PROLONG™ Gold antifade reagent supplemented with DAPI (P36935; INVITROGEN® by THERMO FISHER SCIENTIFIC®) and incubated at RT in the dark overnight, before they were examined with the LEICA® TCS SP8 STED 3× microscope (LEICA® Microsystems GmbH; Wetzlar, Germany).
[0064] Imaging on Leica TCS SP8 STED 3×
[0065] The confocal images were acquired with a LEICA® TCS SP8 STED 3× microscope. A HC PL APO CS2 100×/1.40 oil immersion objective was used for image acquisition. FITC anti-mouse TER-119/Erythroid Cells; Clone: TER-119 was excited at 488 nm (500 nm-571 nm, HyD3), rhodamine was excited at 550 nm (569 nm-650 nm, HyD3) and DAPI was excited at 405 nm (430 nm-470 nm, HyD1). The pinhole was set to 1 AU. The images were acquired in unidirectional confocal mode with 1,000 Hz scan speed (line average 6). The image size was image size was chosen to be 38.75 μm×38.75 μm (1,024×1,024 pixels). The PMT Trans was further activated to enable the acquisition of DIC images.
[0066] Sample Preparation for Electron Microscopy Cryosectioning
[0067] The sample pellet was fixed in 4% paraformaldehyde (PFA) in PBS for 30 minutes at room temperature followed by further fixation in 4% PFA at 4° C. for 1 hour. They were rinsed in PBS and re-suspended in 10% gelatin. Chilled blocks were trimmed and placed in 2.3 M sucrose overnight on a rotor at 4° C. They were transferred to aluminum pins and frozen rapidly in liquid nitrogen. The frozen blocks were cut on a LEICA® Cryo-EMUC6 UltraCut and 65 nm thick sections were collected using the Tokoyasu method (Tokuyasu, 1973) and placed on carbon/formvar-coated grids and floated in a dish of PBS for immunolabeling.
[0068] Immunolabeling of the Ultra-Thin Sections
[0069] Samples were processed according to the method described by Slot and Geuze (Slot and Geuze, Nature Protocols. 2007; 2(10):2480-2491.). The grids were incubated on rabbit anti-BmGPI12 (1:100) or rabbit anti-BmIPA48 (1:100) and their corresponding pre-immune sera (1:500). For secondary antibody, 10 nm Protein A gold (Utrecht Medical Center) was used. All grids were rinsed in PBS, fixed using 1% glutaraldehyde for 5 minutes, rinsed again and transferred to a UA/methylcellulose drop before being collected and dried.
[0070] High Pressure Freezing and Freeze Substitution Epon Section and Labeling
[0071] Samples fixed in 4% PFA were frozen using a LEICA® HMP100 at 2000 psi. The frozen samples were then freeze substituted using a LEICA® Freeze AFS unit starting at −95° C. using 1% osmium tetroxide, 1% glutaraldehyde and 1% water in acetone for 10 h, warmed to −20° C. for 12 hours and then to 4° C. for 2 hours. The samples were well rinsed in 100% acetone and infiltrated with DURCUPAN™ resin (ELECTRON MICROSCOPY SCIENCES®) and baked at 60° C. for 24 hours. Hardened blocks were cut using a LEICA® UltraCut UC7 and 60 nm sections were collected on formvar/carbon coated nickel grids.
[0072] Immuno-Labeling of Resin Sections Grids were placed section side down on drops of 1% hydrogen peroxide for 5 minutes, rinsed and blocked for non-specific binding on 3% bovine serum albumin in PBS containing 1% Triton-X for 30 minutes. Grids were incubated with a primary antibody rabbit anti-BmGPI12 or anti-BmIPA48 1:100 overnight, rinsed in buffer and then incubated with the secondary antibody 10 nm protein A gold (UtrechtUMC) for 30 minutes. The grids were rinsed and fixed using 1% glutaraldehyde for 5 minutes, rinsed well in distilled water, and contrast stained using 2% uranyl acetate and lead citrate. Grids were all viewed in a FEI Tencai Biotwin TEM at 80 kV. Images were taken using Morada CCD and iTEM (OLYMPUS®) software.
[0073] Image Processing
[0074] FIJI (imagej.net/Fiji) and MICROSOFT® POWERPOINT® (MICROSCOFT® Corporation) was used to analyze and prepare raw images of Giemsa smears, EM images and fluorescence images for presentation. Diameter and length of EM structures were determined via the line tool in FIJI. The Huygens Professional Software (Scientific Volume Imaging) was further used to deconvolve the fluorescence images acquired with the LEICA® TCS SP8 STED 3×.
Selected Results
[0075] B. microti major secreted antigen localizes to vesicular structures associated with parasite morphogenesis. The immunodominant BmGPI12 of B. microti is encoded by a member of the bmn multigene family and one of the most highly expressed genes of the parasite during its development within red blood cells. Consistent with the secretion of BmGPI12 from the parasite into the red blood cytoplasm and subsequently into the host environment, immunoblot analyses using anti-BmGPI12 antibodies on blood collected from mice and fractionated to collect plasma (S), erythrocyte cytoplasm (H), and membrane (P) fractions showed the presence of BmGPI12 in all three fractions from animals infected with B. microti strains (LabS1 (
[0076] The localization of BmGPI12 was further examined by confocal microscopy. The analysis identified BmGPI12 in both the cytoplasm and plasma membrane of the parasite as well as in the cytoplasm of the infected erythrocyte in well-defined dendrite-like structures and distinct foci (
[0077] Vesicle-Mediated Export of BmGPI12 Antigen into the Cytoplasm of B. microti-Erythrocytes and Host Plasma
[0078] To investigate at the ultrastructural level the nature of the TOVs, electron microscopy analyses of ultrathin sections of erythrocytes from B. microti-infected mice were conducted. These analyses revealed tubes of connected vesicles as well as individual vesicles in the cytoplasm of infected cells (
[0079] To determine whether the IV and TOV structures identified by electron microscopy are the same BmGPI12-positive structures detected by confocal microscopy, immunoelectron microscopy (IEM) analyses were performed on B. microti-infected murine erythrocytes using anti-BmGPI12 antibodies coupled to 10 nm gold particles. IEM analyses showed that BmGPI12 localizes to the parasite plasma membrane (PPM) as well as to the IVs and the TOVs (
[0080] Export of BmGPI12-Containing Vesicles from B. microti-Infected Erythrocytes
[0081] The finding that B. microti produces IVs and TOVs inside infected erythrocytes and that BmGPI12 is associated with these structures led us to further investigate whether this protein is secreted into the host environment via a vesicle-mediated secretory mechanism or as a free antigen. This was achieved by subjecting plasma samples from B. microti-infected mice to ultracentrifugation at 120,000×g to separate fractions containing membrane-associated structures including vesicles and tubules (Up, ultracentrifuged pellet fraction) from those containing free proteins (Us, ultracentrifuged supernatant fraction) (
[0082] Evidence for Vesicular-Mediated Export of the Immunodominant Antigen BmIPA48 in B. microti
[0083] To determine whether the vesicular system used by B. microti for secretion of BmGPI12 is also used by the parasite to export other antigens, we examined the cellular distribution of another B. microti antigen BmIPA48, which was previously shown to trigger strong IgM and IgG response in infected outbred mice (Silva et al., 2016, Scientific reports, 6:35284). BmIPA48 encodes a 48-kDa antigen with an N-terminal signal peptide but no GPI-anchor motif or transmembrane domains (Silva et al., 2016, Scientific reports, 6:35284). As shown in
[0084] This study reports the first evidence that the human pathogen B. microti employs a novel mechanism to export parasite proteins into the host erythrocyte and subsequently into the erythrocyte environment. This system employs a network of parasite made dendrite-like membrane branches consisting of connected vesicles. We show that at least two immunodominant antigens of B. microti, BmGPI12 and BmIPA48, are exported by the parasite via this mechanism.
[0085] A recent study estimated that ˜398 proteins may be secreted by B. microti during its development within mammalian erythrocytes (Silva et al., 2016, Scientific reports, 6:35284). Some of these proteins may be exported into the erythrocyte cytoplasm or erythrocyte membrane where they may function to modulate the host cell cytoskeleton or to facilitate uptake of nutrients. Others might be further exported into the host plasma to modulate the host response or effect other changes beneficial to the parasite. Both BmGPI12 and BmIPA48 contain an N-terminal signal peptide but lack specific motifs such as the PEXEL motif found in other apicomplexan parasites and associated with secretion of proteins into the host (Cornillot et al., 2016, Transfusion, 56:2085-2099; de Koning-Ward et al., 2016, International journal for parasitology; Lanzer et al., 2006, International journal for parasitology. 36:23-36; Marti et al., 2005, J Cell Biology, 171:587-592; Pelle et al., 2015, Cell Microbiology. 17:1618-1639; Sherling and van Ooij, 2016, FEMS microbiology reviews, 40:701-721). In fact, all predicted secreted proteins of B. microti lack such a motif (Silva et al., 2016, Scientific reports, 6:35284), suggesting that this parasite might have evolved a novel mode of protein export. Interestingly, unlike P. falciparum where the PEXEL motif plays an important role in the trafficking of parasite proteins across the parasitophorous vacuole that separates the parasite from the erythrocyte cytoplasm, B. microti spends most of its intraerythrocytic development without a vacuole, thus eliminating the need for a translocon to export proteins into the host cytoplasm. Consistent with this model, analysis of the B. microti genome shows the lack of orthologs of most components of the malaria translocon (Cornillot et al., 2012, Transfusion, 56:2085-2099; Silva et al., 2016, Scientific reports, 6:35284).
[0086] The electron microscopy analyses of ultrathin sections of B. microti-infected erythrocytes showed that the interlacement of vesicles consists of individual vesicles (IVs) and tubes of vesicles (TOVs) with a diameter of ˜0.110 μm±0.0052 μm. Giemsa staining showed that the TOVs can vary in length between infected erythrocytes with some extensions several μm in diameter. Further analysis of the blood smears showed the presence of TOVs throughout the life cycle of the parasite and suggest that production of filamentous forms represent a distinct morphogenic event in the development of the parasite. Interestingly, analysis of cryosections showed that the IOV system of B. microti is of a composition similar to that of the cytoplasm of the parasite. An enlargement of a section near the parasite plasma membrane in
[0087] This study employed fractionation methods used to characterize the secreted vesicles produced by B. microti. Using this approach, we found that BmGP12 could readily be detected in both the soluble and membrane fractions following centrifugation of plasma samples or culture medium of short-term cultured parasites, whereas BmIPA48 was found primarily in the vesicle-rich membrane fractions. The difference in the distribution of these two proteins could be due to their respective localization in the vesicles and tubules. Immunoelectron microscopy analyses showed that BmGPI12 is primarily found on the parasite plasma membrane and on the membranes of vesicles and tubules. Interestingly, the 10 nm gold particles could be found both inside and outside these vesicles and tubules. It is, therefore, likely that following vesicular release, BmGPI12 exposed outside the vesicles is cleaved and thus found in the soluble fraction whereas the proteins still residing inside the vesicles remain associated with the membrane fraction. On the other hand, immunoelectron microscopy analysis of BmIP48 showed the presence of the protein primarily inside the vesicles, consistent with their exclusive association with the membrane fraction. Interestingly, none of the vesicles could be detected on outer layer of the erythrocyte membrane of infected cells, suggesting that the vesicles are likely released into the host environment following rupture of B. microti-infected erythrocytes as suggested in our model (
Example 2: B. microti Secreted Antigens Identified by NANOTRAP® Proteomic Approach
[0088]
TABLE-US-00001 TABLE 1 List of 15 exported antigens of B. microti identified using a NANOTRAP ® proteomic approach B. microti Gene Annotated protein Protein length BmR1_04g08775 Putative HtpG 712 BmR1_03g03490 Putative EF-1 alpha subunit 447 BmR1_04g05965 Putative Enolase 438 BMR1_03g01010 Putative Ubiquitin family 77 BMR1_03g01340 Putative Ubiquitin C 282 BMR1_03g02390 Putative Actin 376 BMR1_02g03540 Putative RPL18 187 BmR1_04g09800 Multifunctional tryptophan 425 biosynthesis protein BMR1_01g02545 Hsp70 644 BMR1_02g03395 Unknown function 395 BMR1_03g04775 S-adenosylmethionine 404 synthetase BMR1_03g03380 Glutamate dehydrogenase 467 BmR1_04g08050 peptide chain release 433 factor eRF subunit 1 BMR1_02g01430 Unknown function 1094 BmR1_04g08520 Unknown function 1024
Example 3: B. duncani Secreted Antigens Identified by NANOTRAP® Proteomic Approach
[0089]
TABLE-US-00002 TABLE 2 List of 46 exported proteins of B. duncani identified by a NANOTRAP ®-based proteomic approach. B. dunani Gene Protein Length Annotation BdWA1_II1364 229 Hypothetical protein BdWA1_II1520 644 Hsp70 (BdHsp70-1) BdWA1_III2507 1058 Dynamin family, putative BdWA1_I0004 183 Hypothetical protein BdWA1_I0706 376 Peptidylprolyl isomerase, putative BdWA1_II1893 160 Hypothetical protein BdWA1_III3045 132 BdGPI6-Hypothetical protein BdWA1_I0760 188 RPL22p/L17e, putative BdWA1_III2950 490 Protein disulfide isomerase related protein BdWA1_II2303 442 Enolase BdWA1_I0910 177 Hypothetical protein BdWA1_I0555 131 Polyubiquitin, putative BdWA1_II1366 229 Hypothetical protein BdWA1_I0089 797 Metallo-beta-lactamase superfamily, putative BdWA1_III2562 392 Alpha/beta hydrolase family, putative BdWA1_I0812 713 Hsp90 (BdHsp90-1) BdWA1_III2693 126 Hypothetical protein BdWA1_II1642 255 14-3-3 protein, putative BdWA1_II2273 742 RAP domain/Core histone H2A/H2B/H3/H4, putative BdWA1_III2537 400 Rhoptry-associated protein 1 (RAP-1), putative BdWA1_II1831 242 BdGPI8-Hypothetical protein BdWA1_II2038 917 RPL1p/L10e family, putative BdWA1_I0231 618 BdGPI17-Hypothetical protein BdWA1_II1370 266 Hypothetical protein BdWA1_I0253 498 Hypothetical protein BdWA1_III2944 296 phosphatidylinositol-4-phosphate 5-kinase, putative BdWA1_I0708 376 Actin, putative BdWA1_II2269 292 EF-hand domain/EF-hand domain pair/EF hand, putative BdWA1_II2320 476 Sodium: dicarboxylate symporter family, putative BdWA1_III3035 207 Hypothetical protein BdWA1_I0924 299 Ethanolamine-phosphate cytidylyltransferase, putative BdWA1_III2663 516 MAC/Perforin domain containing protein, putative BdWA1_II2261 1416 RNA polymerase Rpb1, domain containing protein BdWA1_III3073 333 PP-loop family, putative BdWA1_II2013 278 5′-3′ exonuclease, putative BdWA1_I0563 825 Hypothetical protein BdWA1_II2256 374 Hypothetical protein BdWA1_II2291 535 Hsp70 (BdHsp70-2) BdWA1_II1496 400 elF4A-3 BdWA1_II2200 108 L7Ae BdWA1_I0250 418 26S protease regulatory subunit 6A BdWA1_II1994 524 GMP synthase BdWA1_III2590 126 GTP-binding nuclear protein Ran BdWA1_I0810 612 Hsp70 (BdHsp70-3) BdWA1_II1634 117 Core histone H2A/H2B/H3/H4, putative BdWA1_II1773 396 26S protease regulatory subunit 4
Example 4. Babesia duncani Secreted Antigens: SEQ ID NOs: 1-46
[0090]
TABLE-US-00003 TABLE 3 B. duncani secreted antigens identified in the pellet fraction after ultracentrifugation of the culture supernatant: SEQ ID NOs: 1-23 SEQ ID Antigen ID Annotated Sequence NO: BdWA1_II1364, MKFLFGFFVILFLRLSKQEELVSLQLGDFHFDFEN 1 hypothetical protein GKYSTHEPFETCFIDVYNYRYDKTGPFLFNIFIKRT LENEYQSLFFKRENGKLVNFAPSHLSSPQTDNTGT YYSTKEPVVLESKNLSDIRNGIKKIGGNKLSSSGKI NWDTISTTLLLKTACGTYSTYSSGYEALLPVKDG NDTFCCCFSKAVLFTGYRFQMKKYEEVKPASNSQ STCKK BdWA1_II1520, MAATAIGIDLGTTYSCVAVYKDNNVEIIPNDQGN 2 heat shock protein RTTPSYVAFTDTERLVGDAAKNQEARNPENTVFD 70 precursor VKRLIGRRFDDPTVQSDMKHWPFKVNAGAGCKP TIEVTFEGQKKTFHPEEISSMVLIKMKEIAEAYLGR PVTDAVITVPAYFNDSQRQATKDAGTIAGLNVMR IINEPTAAAIAYGLDKKGSTEKNILIFDLGGGTFDV SILTIEDGIFEVKATTGDTHLGGEDFDNVLVEHCV RDFMRMNGGKNLATNKRALRRLRTHCERAKRV LSSSTQATIELDSLFEGIDYNTTISRARFEEMCNEK FRSTLIPVEKALRDADMDKRKINEVVLVGGSTRIP KIQQLIKDFFNGKEPSRSINPDEAVAYGAAVQAA VLSGNQSEKIQELLLLDVAPLSLGLETAGGVMTV LIKRNTTIPTKKTQIFTTNEDRQEGVLIQVFEGERA MTKDNNLLGKFHLSGIAPAPRGVPQIEVTFDIDAN G1LNVTAMDKSTGKSEQVTITNDKGRLSQTDIDR MVAEAEKFKEEDERRKCCIESKHKLENYLYSMRS TLNEDAVKQKLSTEELQNGLNTVEEAIKWVENN QLANQDEFEDKLKEVEKACAPLTAKMYQAAGG AGAGGMPGNFGGAAAPPSGGPTVEEVD BdWA1_III2507, MATIRKQRLEDLTDIHKEHLSTAHQLLDVIKSASD 3 Dynamin family PKLIYLHCYQLMKLGGLDAEVPRLVVFGQQSMG KTTLLDFIMGGPIGYSSTDTGTRQPVVIIMRPESAI DPRELELASGSVGSPSSTITSKKIWCKFNGKIMDIH NVQQNMRLHMQSLGERICSEELEVEVYVPDAITA IFVDLPGIKDDSKSGAEFTRRVVRNYVSNNPNDL YLLVKKSSDDPANWPWSLREFITAAAPNGLGLSP QQTMVVGTRAREFLINEKTDIRTQDQLYERVHKR AVLDSKGQALPLHLLELFSLSIQAKDKGDFLTNKE EMKRQIANGQVEVENMIRHGFEESNSINKEGRSV SEELLDTFSIRQFLRSLNSKFSQLLNGHLTNLERRL IRKKIDLERIVASLETKLQCFSPTTVRESIKQFIRQF MEIVHNMMMGNYTIMKLPIPPEQFLGIYGGSLRD NLEDGHELAQNLFPQPDMYESNFYTKITARTEEL YNKKLTMIDTVKPGRYVRYFTSKISVHMFGLIEPP RRVPPAVSGADLGPFDMMGQDYGADELINVEFIQ LNNNEENSLHKDIDRSKLSLLTPLASVSAEHLQVP LYAWHKTKEPKTGWVVVRPIVIDRLPPEILVQKS NYREVDVANKQISFRYLDVEFETKALGSGEEDQL ENSVETEISETPPVVEGVQNRCSNRYLYVTACSEI FLEEHRTAPYYASALEMVSGEHAEANLLNQLAVT NICHWLKFQIKHMEPEHVYSAEVLYQMLRSIDHV VDRADWEPLVADLVQSNVRGTLLHASRLAACAS AAALRRVLRASLAEAFRCIKLTDCDQTLYCLPDS LHFQEQIDHLSEEYCRQKAIDCANAMMNCIIEQT YSIQFDVAVDIFDGCRQFEKYFMGRAGHRSFMGD ALSSVKEDLALRKRRLAMTDIFEKSDAKTSIELIY EEVKVQFWATKLLLSTPLTTKLYTYFIKQVVDKA LPTTHSDPTASIKVEFEQFLIDNILYEQIDGTRTAK SNNRLSADYDLNNKYEQLVQQHNRNKRLLEYISC ALEGISRFKHHATADTDFLAHLD BdWA1_I0004, MISSPPSGFSLHGEDAKGTPRDVERQDSDRLDPG 4 hypothetical protein NFPPDKYWSPSIGLSTIEANRRVVWLLQEAISRYK IHLGYWNTTKSCLTIDILIGVLVLVLFILGAEPNLG VWHVVRPVLLLPWLVLNCYSKITVMRIKSSRACE RVRDVLESFSRELKTGGHTEVEQRRFSFDPPNSLL IPVYRDREWKRLPANVLLAGDVFKLQIGDYFPCN CRIILSCDREGKVQLDANLFNAGSVFKSSHLPSINT NDGGEWTDASFVAVTDSFVQSLETFLSADQGPQS RFMFFNERDWENCQKGPNTPPPTTCVWNFSDYT HFSKYGVDWIQLGIAFFISSIVTVLQIIWSGFQGWR RHVNVFATCIICLCHPAFDPFLNLADIWGNVKLQS LFQWHNEKRFTVDILPQQSSSSSSTFDSGSSEDSV DSEMAASRIPLLHQLRELNRVFKRGLDSEGSLLRT LCSVTLLCFVDDMGLLTEGCATPQELAVVDPSGG IGTQRRQQKESTAMFQSGNIDAIETINSTATSHKD SIGQTEQSDKGTIKAGPREGKQVVRKDPQGGERL VILDVFEDSSQYYKQYVKFNNDAERNCIPQVLSL SFAMSATQFPRVQPSLLQLKAAPDLIHTYMGMLA NGTLHDFTHCLCAFAGSVGLKRSYIRRFRLLRFIV VLDETLGTNGKMLIYFLRDPRKQIVQMLVKAKPE TVFDRSINYHDRARGAILPVSRLTKRKLRDLNMQ WVSSGLTPFAFIYKPIHLDEFNLIMAHLPGVAVFK VGHFLKLDRHQSVKYQESTTCQQDPQADYFGEG GKSDAKARRWYARLTDYDAISMRYGSMHTDLR QRILTNSIGGLVNSCLKNSILLGMCATKYQYPKEV PSRIQSFHEAGIRFVYFSKHDEKQTRIVGGLLGLET SWNSMISLVKSGRYSHVNQDGRVVLPSGIDNIRR HIRDVDDIPLQVSLFCDCTHTSTVEMMRILRENGE RIMCVGNGLRPSNFFVFCEAHSSVSVALGYHPTC RFCRGKRWSGIARAHAFEEATPEMKLSAFLTSLP CDLQTSKMYKVADPYFVMEMLHEVFKEARHMA TNIQDAAAFFKLASHSVAWMLMFQASLGFRRIL MPADLALLIFVYIPLMGSCLLSNVVAEGTMQQMP SRCTKDDAKVTIAAMCKTYPRLLLVATSLLVFYS FVLGQIQALLKIELERLYNFTLDDTQCSRFWRVAS YSCLQEHEQSIALLRTQVQSVIFRENTAAHLAEQT ASFAMAFLYSVSSASWIVRTGRIGAIASELLHSRW IISSAAILTIQGTILVARILAQPVPLASLNAIMPWGL VCGMLLGLSLAILAVDHLHKFYAARQHEMDQKN VESKVTSDLDIMDVTCICEPILSPSTQKISKRTSPPP IKQNVRGILQCKKRSYTSKSNRRRHPAIFNPDTTS AKDMGVLRTRQHYTSILLHFLQRGINGEQLQLVS LDIDQESFRLPLKKHSFGEEAAPAFLDVYVEEDIQ KIPVMFNLVIETKGDYTYRNYFFKRDGDKFINFD VPKLSDYYNVRTEGIATYYLTYKPIRVPREVLKRL RREFPDNNQLKVSNDPEDGPRSNEITLKLKLNSGS LVSYMGTYVPKTSSRGCRSKAPIFNKFRYKLTTD GQAIPVDEVEEEGEEEEEENENEKTEETDDQDRK VVTKEDDEEEEDEEVEEVEEELVEIKDEEEDKCYI EAWEKHSKNYSKVEVATSNKKHISDTGSLKPIDE LSGLFNALERKIATETALESDTTYTPKDNERWIID KCNSEESILDLHIKEDILTFDVPITQLPIQEKEIVDP CNSQRKSAFFNKIGSAGEHYLRAFGSNILSVIDIM KSDLNVSIKKVNSDEINEDLPTKEKLLAMEPIMFN SMDGEDNNNKNTDEEVNGTKTLMHSINSQSIERC EMNSAYNQGSFISIHDGTSDSFMDSDSFSISQKITQ DELLSKHDDKTLLNTDNALNDDSGLKTTEISHTV NDTVIPNLKLEKLLPQSKEQSAESTSSGEVGLIKK NKDVQSTKGKVGDLGNYVQINDDKEHPREIVTV EKFADETGIGTSTKKNEPATTSPLNEFDKIPNNDA AVNDSEKKTPADESRGNTNAKKTSDGNFKTISAN LKPLKPFRPDSLVTAMHISNDNKFLATGNENGEL YVWKFESAKMMSSPEISDNRINALPKMLNWEML SSTPTAGVQAHEFFIYSIHISKIQNRKHSGSVLVVT SGGNNYVRIWALTKSDKENVLILKGDRQFDDDIL TAFQLPISQHIAICGIDQVIEIWKFPPITTSVNDNPK NISSWNKTKERIDVELTIQSVSYSPSGMYIAIGSID GLLSLYSAETMKLISMAICRNEKGWYSNSASITGI VWNTKETLVCATTADSRIRLFSTNIENDNALIYAE KLKGHKYCGENVAARFTGLNDEYVICISKGGYIVI WKHSCTEERIFYDGNPIIKNTNYCKFKIIPKPYIGK LLGVFNPGTWGHLWPTYQEQSVYEPLNDKNSVG CLDRFLTKNTPGVKFSKANPKNAILLVASVDGSQ LLCTIIDASLLVFKKS BdWA1_I0706, MTESVDLTGDGGVLKTIIKPSKFFEQPEQGHEVEV 5 peptidylprolyl HYTGKLDTGFVFDSSHKRQTTFKFVLGDGNVIKG isomerase WEVGVAAMNIGETALLVIKPEYGYGAAGSGSTIP PNATLHFEIELINSRIKPKQKWEMSMDEKIQAAAD AKVEGNAKFSQERILAAITYYEDGISYLSTRDEWP EDAIKASNVTKLQCHLNLANCYIKTEDFANAETH ATEALNIDPVNIKGLYRRGLARVKQGDFKEAIDD LSKLIKIEPTNAAGVSQLKLAKEKYTIFKQRERNV FGSVFKKMNLYDEKSGIRNLDTLPRVFLDISITGH VDVNRLVIALFEDTVPKTVENFKSLCDENAKLTF KGNKFHRLIKGFMIQGGDITNGDGTGGACIYGDQ FDDEGFKDSHSERGLLSMANCGPNTNNSQFFITFV PTKHLDGKHVVFGKIVEGMEFLDILENLSTGEND RPEADVTIEHCGTL BdWA1_II1893, MPSQGTVKAQTGTTEVDNADSSEEESQKSQPESK 6 hypothetical protein AVNGISGTESRNENSQSVDTNSSGDTNPSQSAGGS APTTGDSKNQQGKNVNAESSSNPNSEKSVATQDS STDQTGKTDSSTTHTTTGDSVEQKGDDNTETTDT AQNTATEEATTGSGTEGSADQTE BdWA1_III3045, MARFFSYKKLIAFAIVALASLKEVSFLGGCPYALA 7 hypothetical protein VATTTTTGTNGAATGTNGAATGTNGAGANDTSK NTSDPNTPATPPSSPESNKDNAAGGSDGQKPTGQ DPQKPNAGNGFAATSVIGAATIGLLTLAFN BdWA1_I0760, MTKYSQEPSNLAKSAKAYGAHLRVHFKNTYETG 8 Ribosomal protein RAIQGKMILEAKRYLNDVIEHKRCVPFRKFNGGV L22p/L17e GRCAQAKAFKHTQGRWPEKSCRILLDLLTNLESN AEAKGLDVENMVIENVLVNRAPLGRRRSYRAHG RIIPFLSHPCHVALIAVEKDENVPRFTPEAAKTIKL NKRQIARMRLCNGKGVAK BdWA1_III2950, MISHYCLLLLAAATVFGGEATVTIEDAKSATLEAT 9 protein disulfide TGVPEVAEGDVSIPRGVVTLTADDLHKSIEKHEAI isomerase related MIKFYATWCGHCKILAPEYIKAAKILEEENVNVV protein LAEIDAVAHSDAVAEFEIKGYPTIKFFKRGIPIDYN SDRKAETIASWCKEMLNPALMETTNLEAEIASRK SKIALVAHGCNDKDELCVLFEKLAEVHRMDAHF FSVADSSSVWFEVRHVGDGTLKFNGLSPEELALF VKDETLPLLDEINPANYARYTSSGKSISWLCANTQ DYTKYRSSIVEVAKEMRSHTVFVWLDTEKFSAV NEAFAISKLPAIAHQTMKGRFILSPDAYDFTSKSA MLQFYTDVEQGKIPLSFRSEAEPQDATEGPVMLV VGKTLQQLFTQTDKAVLLMIHAPYCEHCRNFMP VFEDFAKTIDAQAPLIVAKLDGDANESPLDYVSW EAFPTVLLFKAGDKQPIPFKGTRTIEELTSFVQEHV TLAPVKTEL BdWA1_II2303, MVEELDGTKNEYGFCKSKLGANAILVVSMAAAR 10 Enolase, N-terminal AAAAHLNIPLYVHLANLAGKPTNKFILPVPCLNVI domain/Enolase, C- NGGSHAGNMLAMQEFMILPIGAGSFREAIQMGSE terminal TIM barrel VYHTLKKVISSKYGQDATNIGDEGGFAPNIKNAE domain containing EALDLLLEAFRIAGVEGLFKIAMDVAASEFYDKN protein TGMYNLGFKGKEPQNKTGEEMISYYKLLCAKYPI CSIEDPFDQDDFDSYTKLTAAIGEKVQIVGDDLLV TNPKRIEMALGKKACNALLLKVNQIGSVTESIDA CKMAHANKWGVMVSHRSGETEDTFIADLVVAL GTGQIKTGAPCRSERNAKYNQLLRIEEELGNKCE YAGHNFRTCGN BdWA1_I0910, MEEWYTYALRHVDLDLDNFFVEFGETILEDYTRI 11 hypothetical protein YPRSNVFVDNIRKGATLITLSTEHKGLLFVELRYP RPGKDSVMDIIYKNWEDEEVMFSLVWVFGDWVP QNFLYSGILDSGPEYVPVRRHDSR BdWA1_I0555, MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGI 12 polyubiquitin PPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVL RLRGGVIEPSLVILAQKYNCEKMVCRRCYARLPL RATNCRKKRCGRCSQLRPKKKIKGGN BdWA1_II1366, MGNPGLIFIALFKYSFNCYYTFLPFKFDNNHFDVL 13 hypothetical protein PHENFLPRATVPRFVDIYVSADDPRIPILVNFVTGG MTTSGPDTRPTNRNYFFKRDGNKLVNYKFTNKP NDTVENNDIVTRISRERVTYYVGTSNPEDIIVATK DPQNDLNQRENSMYASLATLDPKATIPRIGLKTK SKYNKVALYSDEHKEKIEISNADIDGVMHEKLILF RYRLVKEYRV BdWA1_I0089, MRNVKKRTVPFLWISCFVLYGIYNVEPIALYNKN 14 Metallo-beta- WVHKRVGFIANLKQPPCGITFQGLTHEKAKRGFK lactamase RYAEQSSQVKEASVLFDTLSNTEVLDELDSNDAI superfamily/Beta- QDTPDPLESETIIEVEVEDKGKKTFSDIHNKLKGSI lactamase NKLPSTFAVMKDLVAFLDTMEQQGNEKAKSIAY superfamily domain TSIKKKLITELNKNVEELKSDLPKTKKRLYISPFLK containing protein NTVDPFFEDQYVNPVDFKNYFSLYDNYKQEYRKI AYLCMKTYIARLILGLKRKLKHGIVSTFLWPFAK WAKDNNVRYKEPKPSQLENFERLLKYYGHEFEN VYFEQNIAMVRPPQPKARPPNKWSLIFLGTGSRQP TDTRMTSTMAFTEHDGGRIWLFDCGEGTCACMQ KLNLNPKAVDRIFITHLHGDHCFGLFSFISNSARA LPITVYGPIGISKMLIDIMNFTTTSVLPKFVVHELV LHPDNEKHKTGWHVNYPFGGYIYPQEAGHYLV YENDTCKVMAAPLKHILPTVGYVIKEKSKNENDS TKTQRKIVICQDSCDSSKMVPISMNPNVLIHEATT STTSTIGSSLIMQLVYNFSKGKIDESLLSKINDIISQ EELRRSCFSVTFTTMAMKLRRKCYLIERSYSNLTK TLEQNELKATETESNNADMMTDIYNIVQLVHAAS KIKSCIAEAKKIESQLKELYDAPKHLGPRSTWLKS VYNHVRDVIENNGNTETSSSPTIQAMESLKNLLTK FWTTNKSMGLLFSLEINLPEAAPKDWFSLYSKSV RYSGHSTPWDAGKFAAKINAASLYLTHLSSVSLH VHFY BdWA1_III2562, MLLSCWFNLIACFICICSCYIRLNQTNCTNLRLINT 15 Alpha/beta NNSNIKMKATGKLSMSHFKNKQGLLIRTYAAEV hydrolase family DKPKGSAILVHGNKSHFRADFTNYNVDFYKDKY GLESVDPNIVIREMHAIYPNVDHKIDFNNDYEFHY SKLDGKNALDITPRFTLNGSIVEYLNGLGYSAYGL DLQSQGMSQGHNGHRNYFKKFDDHVVDVIQFIDI IRRNKFHNVNEEWDPNVLGKNYSLNKCFLMGLS MGGNVVLRAAQIFKTLSDFKSNIVDGIVCFAPML DIDMHFSGAFNQLALAIAKMIVTFCHHSTFMINEK YEIDTLNSFLRVNDPYYITKTQTHKAIVSLLEATR TLEKNHSKYPVDMPTLVFHCKDDNVCDFKVFTC NEMI BdWA1_I0812, MADGMNQETYAFNADISQLLSLIINAFYSNKEIFL 16 heat shock protein RELISNASDALEKIRYEAIKDPSITEDQPEYFIKLYA 90 DKNNNTLTIEDSGIGMTKADLINNLGTIAKSGTKA FMEAIQAGTDMSMIGQFGVGFYSAYLVADKVTV VSKNNNDEQYIWESNASGHFTITKDESGEQLKRG TRIILSLKDDQTEYLEERRLKELVKKHSEFIGFPIQ LSVEKTTETEVTDDEAEESADAEGEKDKIQDVTD KDETEAKEGEEGDKDKKKKKRKVQNVTREWEM LNKQKPIWMRSPNEVTNEEYASFYKNLSNDWED HLAVKHFSVEGQLEFRALLFVPKRAPFDMFESRK KKNNIKLYVRRVFIMDDCEELIPEWLGFIKGVVDS EDLPLNISREVLQQNKILKVIRKNLVKKCLELFNE LTEKKDDFKKFYEQFSKNLKLGIHEDNANRTKISE LLRFETTKSGDEAISLRDYVDRMKPEQKYIYYITG ESKQSVANSPFLETLRQRGMEVIYMTDPIDEYAV QQIKEFEGKKLKCCTKENLELDDDEEANKNFEKL KEEMEPLCKLIKEILHDKVEKVTCGRRFTESPCAL VTSEFGWSANMERIMKAQALRDPSITSYMVSKKT MELNPRHAIVRELRQRAESDKTDKTLKDLVWLL YDTALLTSGFNLDAPAEFGNRIYKMIKLGLSLDD DVAEASLDEVPALDEVPVDSRMEEVD BdWA1_III2693, MRTLSRLPPPGDPGHVASFQEIATFNIDEFLANIDK 17 hypothetical protein THDGVSQLQLPPNLLENLKQGAGSLLQHLAEQST GPFATHNQPSLIGASGMAVAAPQMQLPQVAMAP QVMAAPQPGMPPAVAMPQMTSQFAGAPGQALP VAAQAVPNMQAPAGPQLASVPISNVIRGNNIAFP GAVPNEELAMELVMKITWALLMHC BdWA1_II1642, MSEENSQRAQLTYSAKLAEQAERYDEMADAMK 18 14-3-3 protein LLVETCITDKDELTVEERNLLSVAYKNAVGSRRA SWRIVSSVEQKEASKSNSVHKTLAGEYRAKIEKE LNKICLCIIGLLDEKLIPATVDSESHVFYYKMKGD YYRYISEFSCDESKANASASARDSYQKATEIAESE LKSTHPIRLGLALNYSVFFYEILNRPQQACEMAKR AFDDAITEFDSVSEDSYKDSTLIMQLLRDNLTLWC SDVTSDAPDKQKQED BdWA1_II2273, MFGLLRPCLIRLSGAVATRQTFGNLADCLKIAESS 19 RAP domain/Core QSIATTDLYEAFKFISSSGQIRRQAIHDDRFVTLLD histone QLDARISTLNCSYMGNFGIRLGLIIQSLGNLDRED H2A/H2B/H3/H4 PIVEKSVKVIERLCTEMMEKSGNIKEISQLAFAAA SAGLQHKFLDYAKQNLTLNIENADPDVLNLALLA SYKTKVHDKVFLALICEKLSELTDRFTANDVVST LRSLEKTSLMKGFLLRRLSMLIHDNLEQFTNEQLA QCCYRLSILKFQTPVQYSTILSLLEPKFQQLSIHLQI EVLASGCMCQCTDANERLVKLAKSITLTDKVDLA GLVNYIYSCVYLKLYKGDHLTGALEEALARSPFLI RKYALLFKEAYDTLSLECPNLTLELPEAWKMALE NYESAEHDRCIQTSIIAETGNILKTSAGDFETFSKV GPFTVAFADVARKLVILAETPNTLGGLALAQRSIK AMDYKVAIIKYWEWRRLKTEKSELSYAFKRFDK SRLGVLSHVQFVRLLGAIGIHLTRQELKFLQFEEDI RGGFTLEDLEALGRDFYNDEVIATKVLESLQEHF GPCNTLDKQELASVLMKLGASLGVAREELDTFLN FYSAHSNSISVEAFIRGAALGVLELCADHTVTILQ WETSRLQTKTSKMSGRGKGGKGLGKGGAKRHR KVLRDNIQGITKPAIRRLARRGGVKRISGLIYEEVR GVLKVFLENVIRDAVTYTEHARRKTVTAMDIVYS LKRQGRTLYGFGG BdWA1_III2537, MVLSPILQFSFLALPSMLLNNVFALRMSAPTPIESP 20 Rhoptry-associated VTKYGDSLNMLFLDLGNAFHENFSMFQVATSMS protein 1 (RAP-1) NYAETNNDIVSRICERFESQKACFVLASKYINNCA KAKKCMQIEKFHLFQSPDMSIKLMNRAQLAAAIH VFRNSGVYEKNYLKRRFNKIFKRQPFGYSSYRTL LIPLLYSNASFNEYTTFSEIFITYYLNVATFMYATLI YRDTRLARFVNAFDKLNVLLIPLKKHLRNMVTGI ANASPVAFCEEDFEPILRIFGHYLSGFDKSLTPLAN RFAKLIRDVLKNELHKSESCILNRAGDFLKNAGR RAGKAVRDAGATIKERGMATYKGVKGGLAGAS DKIRNRFRSRNGNGDDASSYGLLDEDATGEATDD VKPEDDSTGDNEPKEDEITRL BdWA1_II1831, MNLKWLLGLALIGSKYALGGDPNDSEVDSGKER 21 hypothetical protein GPGKRMTFDELLDELKTAEASVLGIKAEINGGLN RLRYRIGNLDAITKSDYDEISDAIRDIITKRTEFAK AVNKRVQLEAIANKFSERTSMGNLEDIQFSTFWV KLEAITRVPDFQLKEDFVKMKDEIIDVKEKFIEKL KKAREATAEVIPETIVEDQEMKSDLHEEIKSHGDD DIFNDKSDKKQNSGFAATSSSLILLAMATIGYSLF BdWA1_II2038, MAKSALYILDSNVKSKKVDKESLKLIKKQSGKPK 22 Ribosomal protein KILRPVEHKAHNPSCLENAKVELDLDLLKKVIETL L1p/L10e family KKRAEVVRESNTRDLLEDPSRNYVFIQIALTKVVT EVHVKPLQIKLKHPIYTDKEVCIFVKDPQKHWKEI IKRENVPQIKKVIGVTKLKKKYKQFEDRRKLCRSF DLFLCDKAVCCSLPSLLGKVFIQRKKMPVPISMSK GGLGNSMREAIQSTYYKLSTGNTCSVKVGICSMN TEQLIDNIKQVFQTIKKFHTEDPIFRNVISSIFLNWE GTESLMLYSRALADDDIQIPQSHVTSPSKPTAAKP TCKWFGTSQSDLVARIVATAKGGTTSLSVWRQFS KDVIETIDTLNIPDVYRILKCFSILRYRHDPLLNVIS YRIVESLDKIACKNLAEILKAYSKLECRNDFLLKT ALPTVARHLEFFTPSDLSSVFYSYCNLGFHDLNFI RQVEWRIFNTLGKLQSCDFALLFCGLTRLERINTR FVISLACQFCKSLDAIDEKHFSLCVNALGRLEFAE HPHLYGILVQHIYNEIKLHHLTSVSLALLVNGFSR AKPKDLKVFQMLSKQIESRLSEFDIHSLCLITAGY SRISNAMEQVYLFEKIAESVGRKSLQLYPMAITSL MYSFSRAGHVHGPLMFYGSQHLTKFAEHYNIVEL SMVSRAHCLLEIKNDDLMHCIAREIVKRFPNVIPT AADSPRVRRISQDSKDLEHVPQESEKCLILEAGTI NLLWIMQGFASFYIFDGNIRNAIMAICNEMCMRIV DLTPMLVSNFLHALATLRYRHETFLEILVRELEDP RLGVKFNQDELKLCYEALNTFGVHGPIYKVSKM ALKQAHEINGGDSTTTLRQVMAGELKTFQDDSEE LTQELKIPPPPKKKVYIHVPEVIRKHLQVPNAQVT HQYDFVTFQV BdWA1_I0231, MDVFSILLVFSAFYVNAIAADDVKTFLFKKDVES 23 hypothetical protein TVEIDANDDAVLVCPIASVLIIKKARWLPVTGGD MRVKDGFSRTTRIGWLCNGLENCAFRPVAHLSKI GDRYEFLGQPIETDIYKLTVTATCGNFMFKRPGR REMLCIPTSAKPDIVLGCKDNEAIELSYVRVGGKS KHQWRHRDYCAESIIKTAHPLCTGKKTCKIAHDV FLKNAKECIPREFNVEYYCAAPHKNSFYDPLDAV VVDGVSVATKYVLTAEDGARASAKTNAYQVLQ VDSALWESDGATERRDRLELVKFLCDGRAECVFS PTRSIIGPDERKCNDVVFGGMVKDTMSHFMLRAH FSLVPFDPKKYDEKEYHHVTIKSTEKKTLECPVN MSLTFYVALWGGKITDTSPLKGPKHFVEVDINGE KHRYSEIINIVGTQCFGKSKCEIEPLKLKPPRHEKD LKEFPTHEGVKKDDHQLELYYKCIDLQTLPSLVES LISDGPRYPREFITPIQLSPDMRIVVMLDIYGPTVL EVANALKLEIPVARTNEIKISWKDAKISQGIRLVK DTRNYVFEFVIGAEDYIHMTVNSFDNDGSPMSIPV EFEASKRILDFSRGIEDFVVATGEITNFRAFIKS
TABLE-US-00004 TABLE 4 B. duncani secreted antigens identified in the supernatant fraction: SEQ ID NOs: 24- 37 Antigen ID Annotated Sequence SEQ ID NO: BdWA1_II1370, MVTREMYIMGYANVISADIKKFKETLANEITTREE 24 hypothetical protein YEKLKADIASTRAIMGDLKSNVRVLTRLLNEVYIL KTLMREEDVRALGDLSMEAKTLVAGKKELQIKL ENEIREIKKKVDGLPLTKKILEENTEQLLEEISKIE MHVKKSAEAIEKNIDECNDKITQTNVMAEEEHEA LTNKLGITKIVLNGIKTGLLKLISISNRLKAATSGA TREENKILKETILNRIKFILAEGDVVFKRLEKDMT DIEKRIKRIPIEGTLPDLNTHGGYGTIETH BdWA1_I0253, MDFLWLLGFAIIYRKFVVGVGPDEDSDYPEVDVK 25 hypothetical protein SSKVNIGISATVDKFFDDMKLMEEDYKAYQDKIL GALNTVRRRLEKAKHLEATDLSDLANIMLDVNK ELTKMNSCVSRLAALRPYVEKSINLLHEDDKETA KKRLLNFMNPDEMVNVLHMLFSEYKELNVELVH VKKRGTAPSQSESLSKSKIESLSKSLSALNQEPGRI LEPALAIYDKIMNGGTEILEEMNKLNLKIQGKQT MSSMEYLYIVQNMLHAKEYVMESKQPLTSLGYL GTALDQMQFTYSPTEKSHVENIKKEVGEILNGIKD FQNKVKNSIDTVEKRVTTISVTDALPKERALEIISA VPAYVQIFKKQMDELKGAVLNDINELDKQLDEK KRIPSKEHEQMEAKIPILETQVQFFLEFIEAMKYFR VTCEMIPKMMGVDEKKQFRRELILCDMALKNEK QEYDFMIEQFKKVKERIVQTRPRASRFKRKHSGFS TMEPSSLLLVLPVIVSLFY BdWA1_III2944, MGNACCKSSPPAAVSDAKTADKPNFESLSTLSMK 26 phosphatidylinositol- NDGSHRNQEKAPSAKASPRKNVEFKFTDTGYDA 4-phosphate 5- TGAKKWDEKVISALLGGKPTNIEAAPGIDVGDGL kinase VERGPVLLKDGSVYCGQWKGSVRHGRGQFFDV DGTQYIGNFSKGVFEGAGELRTWTGDKYQGLFK NGKYHGKGIFTQKNGDVYEGVFVDGMREGYGTE RYKDGSVYMGEFKGGKRMGNGELKMADGVLYE GEFNDEITGKGKMFWPTGECYVGSFLKGMKHGL GETTWKTGPMKSQRGKYENGKMCGTFENVMRD GKVVKGVYKDGILLQEITDTKPKVAPVVTQPPPV KEQATPTPTPKAAASPTTSTPTRAAASASPTLSRA PSTSSTPATATPPASTPTAAASTQAKPKAKSAKSS SAAKKKTSSKSSAR BdWA1_I0708, MADEEVTALVIDNGSGNVKAGVAGDDAPRCVFP 27 Actin SIVGRPKNPALMVGMDEKDTYVGDEAQSKRGIL TLKYPIEHGIVTNWEDMEKIWHHTFYNELRMAPE EHPVLLTEAPMNPKANREKMTTIMFETHNVPAM YVAIQAVLSLYSSGRTTGIVLDSGDGVTHTVPIYE GYALPHAMMRLDLAGRDLTDFMQKILAERGFSF TTTAEREIVRDIKEKLCYVALDFEEEMTNAESSSEI EKSYELPDGNIITVGNERFRCPEVLFQPSFIGMEAA GIHTTTFKSITKCDVDIRKDLYANVVLSGGTTMYE GIGQRMTKELNALVPSTMKIKVVAPPERKYSVWI GGSILSSLSTFQQMWITKEEFDESGPNIVHRKCF BdWA1_II2269, MAIWKLFVFGICGAGKVLGYRLDDVLQGDGEDF 28 EF-hand ALFGLGKEVIEARMDKLFSVIDLNNDGILDLEELA domain/EF-hand AFHAKTFQTILDLQLNHEMELVDRNKDGFVDVEE domain pair/EF LKVAFEREGTQDVDISTVEKGLQRRFVAADKDQ hand DGKLNRQELGLLLNPGRDEELINIEIEEIMQTYDQ NGDGLVSLEEYSHGRSDQEGVSAEFKPFDSNADG FLSREEIRGVYVEENKNDLDSEHEDLFAITGKKPIT REVWNANLNKIAHTSLTDHGEMLRFPEDYHMDL GDIPRDRKDGAEERPSGEL BdWA1_II2320, MCGNGIINNLVYLKMDSIRESTSFHNDFYYITCPF 29 Sodium: dicarboxylate SIKGFLKEYWLSTITFFLTFAAVFIPSVFKLDLKDH symporter family ADYVMLPANMFLRHIRGFVVLFMFFASAAKIRLF LQRKVDSLKTRIMIKYLIAGLLSLLVTLGLAALIIP LNTSLGGNTYFSPHSIKDYNPTTEFKNFINHLAVH DLPLNMATTRVDFVNEFGKDQLHEGLGDGYNAP GFVVYGLLFAFAIYTMDEHTDALCNIITAMHKCL LSVYWILVVYSPFAFFLAGLVTFDELKVKGGLGIA LMGYLYLTLAVLAVFVAWSFIVVPLLHFIRTARN PYPTIIKLLPYLPTAFCSGSSVLSGEKTKDFLKKRG FDPDDVENYLTFSTLVNFSGTTSGFTVCAILMLKL FGKSLDWVTVLKIIVTGLLVGFTIVEYIQGYLFGII FFLNNTMLPPGSIILLLQIDWLLDRFRIVSNVIDDA LTIDEITNVKSKLSSCPFHKV BdWA1_III3035, MGEGTNKTQYPFILGLHEEVVCAAKRLEICLLEKI 30 hypothetical protein EQLGHVDDKGWKVCVDSTIEGKLLGNHITCLSLC KVPNGSHGSEFLLAVGYKSGLIALARVDSQNEFHI LSHNDQNKSSISQIELKYVGESTAAYNLMSLRVY AFLDSGKIITFSWGDKIQTSIQSGEHVLGFAVNSQ NTTIAILQNTGIVCRSLDTETILRTLENVQIVNANR HYLSWHPYKNLLVFLDNKGISYTCHPKWDVYKF SQNSQHQELIRHVQFGICGDFVLLLTASFDKIIIWD FETESILYSHKGCDMVACGLIHLSNKRVRLAVFA NFASNEFWRCKRLIMSGDELMEEAKSELPQSHKT RRLKRHITELDDYHIRKMVDQEAVDEDNEEQDEE YKDEETYRDILDSYDNLDKDEMTHFDQSRVHVM HEISKLRKKVASLDNRVAERTTLVPGSCPAPDDA AVQWVLFWDEVGQITKQLISDVWCLHVHVFSGP LAGYKRKPDRYNCHTAALNQKYVVTGSEINVDG GHVHGILTFLDFENNTQWDRRFFNEYISAVAVGD SFVAAITADGILYILSLARSLMGVFQLKGSPIAIAA RGNVLATITEATTLTNVSSFGHVRMFWVNGLRGL AKNPASRIVDLYSDSLVLGPDKAISWISFSSQCTL WITDTSGQLMALLPAIESCYKVGGYSLEWIPFMN LENLVNESSDYEPSHTVFPLYVSDMKLNYILLKR GQSYPTCSQPLNFMGYTLKKACLRIDGCVGAYLP FQKFNGLVTKDPQLREVIGSDIISDVASIPWQQYD EMRHIQSLQACQNEYILKVFQNYNFWMQNSQGIS DDAISAFGNAERVHDKWTLRILRKTKDSKQDSGV LLDALWMLRFPKCLEAAFSILQNQLDAKQRQVL QEASLLLENVTPFENNVQPLDAQSQPTATVSIPKQ DLDPIPKKHAPLIQGPSNRLQEFVPLGNEPEESGPL FKNILE BdWA1_I0924, MVDFEPESCRKHVDKNTRIYVDGAFDLLHWGHL 31 ethanolamine- NALRQSYKLGGELIVGINGDVETFHAKGISPIYNQ phosphate DERAELVKGCRWVNEVMVGTPYEVNLDFLVNIA cytidylyltransferase KCDYIAHGDDIAIGASGKDAYDEPKKAGKFIFFRR SLGVSTSTTVGRLIDALESDHFSHLSKNSENKLQY GDFEKALQENEEQMINEGIIDDALFSGNNKHDNK NKKSTDVFPYPRFRLSTSLLGEFITPKPKPKGGKII YVDGSFDVFHVGHLRLLKRAREMGDYLIVGIYD DQTVRTLKGTPFPFSSLMNRALTILGMRYTDDVV LGAPYVPSRTYLENLGITTVVTGKQHDSKMINRD FDPYREARDMDILVEIDSGSDITTSDIIARVSSRMD QITANIRKRCAIEKTRKNIVCSL BdWA1_III2663, MLNNANTVKEVGFRKSVIYQPNVFKDADGVIKIK 32 MAC/Perforin TNNKNIWIRQNSKCWEFTSKTPITSVDDYYKELLS domain containing DFNTDDSTSITLYNANVDLAKRGFNNFHEGYTKI protein KKLYCSILEAGLMIPYSGMLSGDFMYAVVSLPDK LDITYCPIDNYMENPTKSECENINRWMEFFKMYG THVSTHIITGGKIFHEYKTLNITEYRKKSKFRQSTN IFEVTNINFDSAGQKSTKNTIVFGGNYVKGMESEA RHFYNEWSKTLESRSLPIKVTVKPLSIFMSYRNDI YKEALKFYRDVALLTTVGIQYSTKIDELLRESTTV VSDDGMAHCPTNQIVLAGFIMSKNPKVPIVNCEQ GKILCSNGTDKPASVYIICVKELNDIITTSTSMENV HICPNGNVTALGFAFRKQSESDDWTVVIPRIGKQ QMINYTKGLNMSWLLCVPIEIMFWHMEMIIDSAP KDDSRTVSCKTGWTILKGFKLMFLKKEDTTRVVL EECISHQQHCILNCNEECTKMYGTILCKKQHD BdWA1_II2261, MSTNGTVRWRTSSRSQLRDRRKNALTRRLRFEV 33 RNA polymerase GRLDTEAFETRTKYLREIFLDVDKRNSHDWTALE Rpb1, domain KKILLLDWCQNPQKFEKWKPAATEDMQSRFFAN containing protein LIFNYSHLAMINSGMCIVIVRLVPLVVNIKAPYQC VILLVRDNVNGMVIDRLKVGLIWRGKDTNTDVL GKSTRIFTPPLSTDFQFLSHGIGYNFKSLLPQTEFEP HVACSDCISRELESLCTLETPDIVLESPALGMEVTS SFVNGHKVYLVVTRGVDGIVRIHRVQELIQTSIDII RTWEQLRLKHLKRTMQSQQYAACTGCLVHKEEL QQLYHLKQCARSFAPECGPMQFVGLEMIDVARP RTCATKREACGLPLALGYTSVRNEQAVIPSLLMA MASNTVCIWSIYSYLKEWNLNPLGPIKVMTYPDG TLPTDISSTVSIAASLEALERLEFIHAPKFYTTDDR GHLSLWSLGSTGPEAQVTLDQNALCSCAVNRSYP HIVAVGLDVGKIKIYNTLTQGTTCIAQMEHPLLTK VQTLQSFLPDNVYDYQRWYHPVVKLEWISDVFIL AQYSEPIFTTESTNASAVAIWNVVKDIFDRDDALV CNKHWSLNSCNLYNTWHLASKLVCLYGGHFGCI CGVLSSDAKWSAEQGLLAVTIDSTGQLHVFKPGI WTWGDCDDPMAIARLTGDCEFYHSILARVQEQH RRLSVPVMEIDNSVDMGQGTRLRKTRAKFSTYIQ DAQTQLESVETASGLEEINSLEKLPMFCKKTLRLN SESEKYLKLVHRLVAEREHAEFLLSQGTPGKTGK RKCPLGAPNIKQEPLELIEAPQGPSSEAPVKSKNE YQNDAMEPESFNFSSVLTKFDTDVSMEPETELVE TALPVVQPTKKRLVEKGLEACCIEGVQFDIMGDV EIARCAELEVQKRELYYYLSSMPFPQGVLDLRLG CNRNDTKCETCGRALMECVGHWGYINLQLPVFH VGFFKYTIQLLYCICKRCSSLLLPFDTVAQLRDAR LRRSDDPLARAVIFKRILSSCRKVTKCPACGARQG VIRRIVKPTMDQFMKLRHVVKYKEGGKIVIVEDE LNPLSVLRLFEAIDPVHARILNIMDPQRLIISNLPVP PACIRPSVSLQGQGTTEDDLTCILSDIVELNNVMA TQMQQGFQTNQVIGNWQFLQLQCTRLINADAPA VSQLLAAKHISKPGRGICQRLKGKEGRFRGNLSG KRVDFSARTVISPDPNIGIDEIVIPEYIARRLTFPEK VTSANLGVLQKAVLNGVSKWPGACYVMKRDGV KCTLRFANPKQVAESLQIGDIVERHLWNGDVVLF NRQPSLHRMSIMAHKARVMPGSTFRFNECVCNP YNADFDGDEMNLHMPQTYEARAEALHLMGVLQ NITTPRNGDPLIAATQDFLSASYLLTSKDRFLSHQ EFCQLLCYVGDGILHAQVPAPAIVYPTFLWTGKQ VYTAILRQIGAVVNLECREREFQHPEPNLFGRMQ PNFMCIKDGHVIISNSELLCGALAKKTLGASKDGL FYQLLRRHGPHKAADVMLKVSKLTSRWVSDFG MTIGLDDTTPSPMLLATKQQLLSDGYAKVELAIA NAANIEPFPGCTRKETLELQVKGILDDLRNQAGK ACNKSLSANNKPMIMFNSGAKGALINIAQMIACV GQQNVMGQRIHHGFIGRTLPHFEVGCIDAKSRGF VSNSFFSGLDPAEFWFHTMSGREGLIDTAVKTSET GYMQRRLMKALEDLAICYDYTVRTSDGQIVQFIY GDDGLGASGAHATTPKALQETLAHVLTLSRFQRT PTILNSRAPGPNSKGDLRLPLPSGDGDDENFECGI VPKCKLAMPPKVKAKASARASWRTSQVGTPLDE ATSAQINRNMYAHWVVHVHLDPEHAKAPLFGDE VLDWVALLEPLCREPLPRCMQESVTSMDHTSEPR IHASSMEIDKRVNGCSMKATNTGPRFSAVLLDTIR QWAARLQTLDPEIQREYESSGMDLVQFRAFKIPP QDRISVLQIYEFLRCAWSDYLHGICEPGEAIGALG AQSIGEPGTQMTLKTFHFAGVASMNVTLGVPRIK EIINAASVIQTPIIEVPLAKKDSYDFAKSVRAKIER HTLEQVVHSIKQVYTPGATLLLVSLDSQYIQQNM LQLDASSVCAVIRNSNLITKFKLSKHNIEAPQKWL VSIRLVASEALLFQLNALVAALGQLVVCGVKDIK RCVVKREQKDVINSLTPGATFEYALAVEGYGLQQ VLGIFGVDAHRVISNHVAEVAKVLGIEAARLVIVT EIKKSMDAYGIDIDGRYMKLLGDVMTFRGEVVGI NRFGIQKMRASSLMLASFEETNEHLFQAAVHGRR DPIKGVSECIIMGKHIALGTGAFDLLYRE BdWA1_III3073, MANGGLYVFPLLLCMHSVYALQCPHAHRACFLK 34 PP-loop family YNGIYTSKANKLGVLDVVNVEDNALDLEDVVPL QEVYKFFNNYLMPFYHKRVSQNYGTNVPIIISCSG GVDSMALLHTFGLIKENSHTFIKKHPINYIDGSNA VIAETASNVLKYIFENVNVIYFDHKVRSDTKVDIEI IENACKKYNFNFNVQELDCNDSYFSNLEGGFQAN SRKWRRRELKKYVMELHSSKMLSNEGKNKIGIR NGSYNSITTNKQDGLNSNSIGIVFMGHHANDNIET FFMKFIRGTHLLQMSEINDQSFLDSKDRIPLIRPFIH LPKNALHQFMQDFRFQYNEDSTNVLFDYGRNQF RKLVMPNLIEYIMSINKCQNDKAIDLLDKRIHVLS KQASNFRNDIEFQIQMFKVYLKSKYGDLLPIKKK RYMNRMGEIYSDFYKRLYFNRYNTAIHSNIQELK YFHDLNIPIMDLFFVDEWLILESKLLREEVLYDFFS HHFRKPLFYNAFTKIVDRLEVNFSEGSIKQYCLSK DVSMSHQGSLLKVKHRPEKNEKVLIFKDDLCSFS VLDNLQLFVEKAEGYTRNVFHLLFKVPMYATTES IDFDIRLFRNDDVLPSKILWNREAGSLLTHMKIKHI IKDYIPVIAMAGTNKIVGFYGFNIIPPYHCRGRQEV TYSFVDVAHGSTEYREYSIRVTR BdWA1_II2013, 5′- MGFFIQSQLLSTLWILIVIPQNVQCLRNNSRNNSL 35 3′ exonuclease AFASGYAHSNVRNSLDSLNGLPNLINRKEPTCQD GFKLFGVPRMYGWMIENLGKINQSFDTCDFYED VDYFYIDMNAVIHSATHGNMSPIVEIEDEQRMRRI TSTLLKLFHMIKPKKVMYLAVDGVCPSAKINQQR TRRFRLAKKVEDLTARVQDICEMKPIEDYNIDKLP CGSYDNVTFNPNYISPGTEFMQMFDSEIKNWLAI KTLEKQWGDCLVIYDGANIPGEGEQKIYEFMRKL NESKVKSRNKNHLVYGLDADIMMLSLLTKMPNV CVLREKRDYTPHILSKIKPEPYFTPETGIVHYHAN DYIDLEAKHFDVVSMMDIRRALYNRCIKYVGLK YDLANIPFLNQENVPNRLADDFVLLSFLAGNDFLP HLPTVDLEFHTFSDMLNSYFFMLPRLHGFITRGYR IHMGRLQKLFRVLQRQEVHVFKEKAQHESVPEY RDVTKYAEHYYRVKCNINYKNRNQVTRMCQEYI KGLVWNLYYYYKGCPSWNWCYKYHYAPLVSDL SQTSGVFVSFKRGRPIKPLEHLLAVSPPNGNELLP EQYRKLSASPNGELAEFFPTDYEICEDGKVNEWE HVVKLPFLNTNKLVTHAEQANQHLKYNSLSKNK LGRVNVYKCRPHNVNKSGDNRLIFDDLTKKGLIV RDGMHYGATFSVYEERPGLAHSSCLIFARHGQTPI MIKDLVRWTRISQAANKRVLIITILYKTMNTSGWS SDFSSLSNSDKITGKDRINEQESISVPSGLHCGPAG RRTLIKWIEENYRVRKGMDDTNPGVVACRLWQD SQGFSDPVSKAAIDLLRLQGIPANVTYKTLFEQHL SKMLSSLINQSRNSQARLYSIAEKMIGMFQVAEIQ PLICDVLDQLDEIPQFALPKLLDDAASANNFYKIC NDGLKRKIWATSAVRLYEEMLPLFQEAVLCIQLG VLEPRMHHASFIQSCRERYEIVIEQICQMIGDCKS QASRQVFRLTMRIIKVLYVKSILGDKGDFQIYFTP HDHLDTALKLNWRDLAARSVGICFKSLQLEELDE AHDSFSSTDHSSFCALAHSIISRHQDMHKITSSDM QDLESFYTLWSLVDTVIASPCMSLDNEQIASVLN VVLGDLRIRDEIDLFDASFVLGHFEFLIKISKYIVT RSININEQLPEDQDTLGQWFSLASLGLSCGYIGAC QFLFRAQWNNDTCIWHFPTETEPGKPISLSKLLGY TFPYNRSDQMCLDTLPPFFYKIVHGQASPHLSETS PVRNPYEANKASLRNLLCLYLPMVDVWKFKRA MMDLKAFEQNGYGTLLNSFKRQTFQTEKLLFYM QDMAIRDFESDDYDWTIARKLYQNLERLQALKL ATPPHGTVMDPKSPQPHVDDGKRLMGTILIQGDI TCGFIRMMGVSMVEASDEMARRLENYWKNLLQ PYMCCMQFRLLRMLFSNWTKISSFSLDVLCKMV KGYPIVAFLPLFTKAPNCDISTALGRLSASLQGTLP NHSSKGWHRIVFTMYNKHS BdWA1_I0563, MKLQINSFNHIFFIKQNIRTNLFKLSQKPHPFNLKF 36 hypothetical protein FRQYSHHEIFTSIKLASNKSEMASVTQILLNRSNY KGVVTLDNSTVVSANDIVLYSEFNSKISFSLTLDT LKKLTKLQTPCKVVWNYVIDSILSDSSHFTPNEIA KILVYILNIKFNDIFIKSKIELASETLIEKLNGSLSLM VGFSVIDIKRAGLRNISDTIRHDAEFDKLDSMDFEI IWKLWYTLSKQRGNERIRGLLFEYLERGKDKVYK FDLPKVIRSMDIILSNSKSLDLLELKFLEKLFKRFL NLYNSDNIPTFGGSSNIYMTTIQAKKALYILGNICK RNPKIFENIVKHECAMLSLVNFLKQVKEITSRSLE VHCRLYNHLKNINVENTNMKHNVRIEAIKRSSNV HAKMFVYCVEFLNNFDLITKANYNNASSYSKATS DNKRSTDLDHTIFDICTNLLRCTAQSSFLIHNVTM YCRSIYQLLVLALYNTCTGINESFVKAALSSISNIQ ATTLQMDRDNWDISSVIRNLSYIDKGISVLADISY SSIILEPIQNANAEIVSKCSSKYEEVLMGGEILQLD KLVYCTLLNKYNIIDGTMLDVLLQFIKANELTSRE LCMFCDLLLKTREMLMDDCDKRNNVLAKLLPLA LELLKSIDFDFNKRTLKDLLLILECLGAFGFSCAD DLSKRILEHGAKLTISQLMKLYSIVNDDYKRRIRL LLPGILKQRGKINITAILKVLNVLDIDYNLLETLEH NLGDDLYLVDKAYFARKRLLHQRNSSNPRKKTK VDYNNSGSHHMFIIDSSVAKAFIKCNKGTHSSSKL YKILVSKYQSSSSSMAL BdWA1_II2256, MQGGWLLVCIVGFLACFGASTKPNDKKATNEEL 37 hypothetical protein TCPAVNDLQGTPIDLQYTFDRLDMTSLSRLLAIQN MYSKRNPRNIRIPKFWNTDVAHGIWSRFRIYGTN LVYKTSRGDGSCLFWSVSDSLRLGGFTIKKIKENL DHYGTKNRGIYEAIMSLPNDDEYLTMQDIQRIATI GFVGFDPDVEASVKQWDREPILSHLETVKSLKTA YMGDVIPFDLGCHRFTSFRAVDDFYNGIANDATC IKAGRDLFNHTIKYLASGAWGYEIDIYAIETALNL KIVLISYNTGSLVCYYWSEDYVPQSMILLYYHDS RHFDVAGLVDMFKVPNVRNPRAKVLTSFHIREM PMALAIILKDDCRITKRNERFSFGNEDLTHLY
TABLE-US-00005 TABLE 5 B. duncani secreted antigens identified in the hemolysate fraction: SEQ ID NOs: 38- 46 Antigen ID Annotated Sequence SEQ ID NO: BdWA1_II2291, MQMFNRFLKASVALLAVASFGIQYIFAKGSNSGK 38 heat shock protein IEGPIIGIDLGTTYSCVGIYKNGRVEIIANEMGNRIT 70 precursor PSYVSFVEGTQKVGEAAKSEATINTESTVFDVKR LIGRKFTDRDVQEDMKLLPYKIINKSTRPYISLHD GKEQRTFAPEEISAMVLKKMKQVAESYLGKEVK KAIITVPAYFNDSQRQSTKDAGAIAGLDVVRIINEP TAAAIAYGLDKANAESNILVYDLGGGTFDVSVLT LDSGVFEVIATGGDTHLGGEDFDRRVMDHFIDIF KKKHKVNIRDNKQSLQKLRKEVEAAKRTLSSTTE VLVEVENLINGIDFSEKLTRAKFESLNAELFEKTL ATVKKVVEDADIPIRDINQVVLVGGSTRIPRIREMI KEYFGKEPDYGINPDEAVAFGAAMQGGILSGESS DNLLLLDVCPLSLGIETLGEVMSVIIPRNTMIPAHK SQVFSTSVDNQPMVTIKVYQGERKLTKDNVILGK FDLSGIPPAPRGVPQIEVTFDIDTNGILSVSAEEKG SGNKHNIVITPDKGRLSPEEIERMIKDAEMNAEKD KEVFNRVQARQALEGYIDSMTKTINDDKTGKKLE DDEKEKIRDALDEGTKWLASNPEVGADEISAKQH EIEAICNPIISKLYGSGEDSDDSGYSDEL BdWA1_II1496, MAIPDNNNNTQSNGFDTLESNYDEVVDSFEALKL 39 eukaryotic initiation NEDLLRGIYSYGFERPSAIQQRGIKPIIENYDTIGQ factor 4A-3 (eIF4A- AQSGTGKTATFSIAALQIINYNIMSCQTLILAPTRE 3) LAQQIQKVVLALGDYLKVQCHACVGGTVVRDDI HKLKAGVHMVVGTPGRVYDMIDKKALLTDKIRL FILDEADEMLSRGFKGQIHEVFKRMPPDVQVALF SATMPNEILELTTKFMRSPKLILVKKDELTLEGIK QFFVMIDKEDYKFDTLCDLYESVTITQAIIYCNTR RKVDMLTNKMQEKDFTVSSMHGDMGQKERDLI MREFRSGSTRVLITTDLLARGIDVQQVSLVINYDL PMSPDNYIHRIGRSGRFGRKGVAINFLTPLDMDA MKSIENYYNTQIEEMPADIAAYM BdWA1_II2200, MSKKLKTKGPENINHSLQLVMKSGKVCLGFKSTR 40 ribosomal protein AALRSGKAWMIILSNNIPALRRSEIEYYAMLAKCS L7Ae containing VYRYSGDNNDLGTACGKYFRVGCMAVLDAGDS protein DILRNIE BdWA1_I0250, MTSVNSDVDISEVSQMSDADIRVRINLIDSEIKILR 41 26S protease SEHTRLKSRQKTLQDRIKDNLEKIQLNKQLPYLV regulatory subunit ANVVELLDFTSDDEQDDGLTPSPSQKKSKSLVIKT 6A STRQTIFLPVIGLIPASELHPGELVGVNKDSYLVLD KLPPEYDNRVKAMEVCEKPIEDYSDVGGLDKQIQ ELVEAIVLPITHQERFKKIGIKPPKGVLMHGPPGTG KTLLARACAAQTKATFLKLAGPQLVQMFIGDGA KMVRDAFSLAKEKAPTIIFIDEIDAIGTKRFDSELS GDREVQRTMLELLNQLDGFASDDRVKVIAATNR PDTLDPALLRSGRLDRKIELPHPNEQARCHILQIHS RRMNVNPDVNFKELARSTDDFNGAQLKAVCIEA GMVALRRDASELEHEDFVEGISMVQAKKKNTLN YLN BdWA1_II1994, MAGHAALILNFGSCFSGVLVRVLRDVGINCVLES 42 GMP synthase AEKALDALNTNSTVKVAILCGGLDSVYDETSLTV PEEFIKACEEKNVKILAISHAFYALCKTLGARLMN GKGNDYIIDTVTVQRPMVLFNNVGKHFKAKINPI NGVEVLPAGFESLATFGNGHYAAIGDEKRGIFGV AFHPESDDTENGLVILKNFCLEQGACPIEWSMEQ YLKDELARCIAQCGDTKVVVAGLSGGVDSTVCA AIVHKAIGNRFHGVMINTGLMRLDETKKCAERLK KEIPGIQLHIRESADVFFGELKGILDPEQKRKIIGKV YIDEFERAIKDLGFDKSNCLLLQGTIYPDILESELN RRNQMPIKSHHNVGGLPKDLALELIEPVRLLFKEE VRKLGRLLGLSQESCERQPFPGPGLGVRVIGELNP RNLDLVRRADAWNQILDARGYRSKISQSGCILL ADVHNTGIRNSGRTYGHAVIIRIIITTDFVTAQWA RIIDTDCLAEISKTITDTVPEITRVCYDITDKPPACIE WE BdWA1_III2590, MAEEMPQFKLLLVGDGGVGKTTLVKRHLTGEFE 43 GTP-binding KKYIPTLGVEVHPLKFRTNCGGIQFNAWDTAGQE nuclear protein Ran KYGGLRDGYYIKGECAIIMFDVTSRITYRNVPNW HRDIVRVCENIPMVLVGNKADVKERQVKAGHIQF HRKRNLQYYDLSARSNFNFERPFLWLSRRLLNQP QLVFVGECAKAPEIQIDPLLAQQSERDLEAAARV AIDDDGDL BdWA1_I0810, MADRFTGRNNREAVVAYPGWFSETQKQCLRAC 44 Hsp70 protein VTASGLSCLRVISHVHAMAMDYGVYRVKQLNDE TPTRVALVMIGHCHASAAIVDFYASHCSILSQVSR RNLGGRNLDMMLMKYMATEFSKKYHCDPLENN KTRLKVEAVAVKTRRVLSANAESSYSAECLMED NDMSGHITRTQFEEMCNAEFIPQLIEMLKECIEAS RTDLDSIFSVEIAGGSSRIPCIQQAISSIFNKVPSRTL NADECIARGCVLEAAIKSNHYRVREYKTRLTLPR SLTLGYFNGQEPMLLEAIAAGTPLGDPIRVTLQAQ APVCVRVALGDALDPRSQDALGTLDIARHISQEA QPAPVTTNDGAAIQTDEQDAEIQSESSPSGGISVTL GFDDCGQFVASPECCEYRWLPATILDIARLEAAEL EARGRDLKENSRLQALNDFETLLYTVRDKMQSS HRDFIDPQMIPAYESELDHWREWLYENSGASQET LQEGIDKVSSEWKRIDKYFKEHQNKLENLEPFLQ RLQERYNFCCEDNNPNWHGATPEERLNFAQELM DLDSRVRQMHQDESQRPRHMEPLFTMQQIQGEM QKLLVSISEFCQAKAAKAPAQEPPEQQPKEQQE BdWA1_II1634, MDAGGKIGGKIGGKVGGMGKGGKGKTGSGKGK 45 Core histone KAPMSRAARAGLQFPVGRVHRMLKSRISADGRV H2A/H2B/H3/H4 GSTAAVYASAILEYLTAEVLELAGNASKDLKVKR ITPRHLQLAIRGDEELDTLIKATIAGGGVIPHIHKA LMNKGPAQVLVKPPKRI BdWA1_II1773, MGIVTASIAPLHIGDDLYTRMKTLEKKLEICEIQE 46 26S protease NYVREEYRNLKLELIRAREEIKRIQSVPLVIGQFLD regulatory subunit 4 MIDKNYGIVSSTAGSNYYVRILSTINRELLTPNSSV ALHRHSHSVVDLLPPEADSSIQLMQVSEKPDVTY ADIGGLDIQKQEIREAVELPLTCPELYRQIGIDPPV GVLLYGPPGTGKTMLAKAVANQTDAKFIRVVGS EFVQKYLGEGPRMVRDIFRLARENAPAILFIDEVD AIATKRFDAQTGADREVQRILLELLNQMDGFDQN ATVKVIMATNRADTLDPALLRPGRLDRKIEFPLPD RRQRRLIFQTITSNMNLAADVDLETFVARPEKVS AADIAAICQEAGIQAIRKNRYVVTTKDFERGWKR HIRKHERDYGFYGV
Example 5. Babesia microti Secreted Antigens: SEQ ID NOs: 47-63
[0091]
TABLE-US-00006 TABLE 6 B. microti secreted antigens identified in the supernatant fraction: SEQ ID NOs: 47- 54 Antigen ID Annotated Sequence SEQ ID NO: BmR1_04g08775 MSSQETFEFNADISQLLSLIINAFYSNKEIFLRELIS 47 NASDALEKIRYELLRDGTKVSDESEFSIKISADKS NNTLTIEDSGIGMTKADLINNLGTIAKSGTKAFME AMQSGCDMSMIGQFGVGFYSAYLVAEKVTVVSK HNSDEQYIWESSASGVFTITKDETTEKMKRGTRLI LQLKEDQTEYLEERRLKELVKKHSEFISFPIHLLCE KTKEEEVTASDDEGDKKEDDKKEDDEKEDDKKG EDEKVEDVSEDKKKTKKVSTVTKEWEVLNKQKP IWMRQPNEVTNEEYANFYKNLTNDWEDHLAVK HFSVEGQLEFRAILFIPKRAPFDMFENRKKKNNIK LYVRRVFIMDDCEELIPEWLSFVKGVVDSEDLPL NISRETLQQNKILKVIRKNLVKKCLELFSELTEKK DDFKKFYEQFNKNLKLGIHEDSANRNKISELLRFE TTKSGDEAISLREYVDRMKPNQKYIYYITGESIQA VSNAPFLEKLKDKNIEVIYMTDPIDEYAVQQIKEF DGKKLRCCTKEGLDIDDEKDEEEEKRFEQVKQE MEPLCKTIKEVLHDKVEKVTCGKRFTTSPLALVT SEFGWSANMERIMRAQALRNSSITSYMVSKKTM EINPYHSIMKALKERVAADKSDKTVKDLIWLLYE SALLISGFNLEEPTQFGNRIFRMIKLGLALEDDQPD DTDLPPLDEGVAVDGGDSKMEEVD BmR1_03g03490 MPKEKTHINLVVIGHVDSGKSTTTGHLIYKLGGID 48 KRTIEKFEKDSSEMGKSSFKYAWVLDKLKSERER GITIDITLWKFETQKYEYTVIDAPGHRDFIKNMITG TSQADVAMLVVPAESGGFEAAFSKEGQTREHAL LAFTLGVKQMIVAINKMDSCQYKEDRYMEIFKEV QQYLKKVGYKVESVPFVAISGFHGDNMVEKSTN MPWYKGKTLVEALDQMEPPKRPVEKPLRLPLQS VYKIGGIGTVPVGRVETGQLKAGMIITFAPTGLTT ECKSVEMHHEVVEVASPGDNVGFNVKNVSVKDI KRGNVASDSKNDPAKEATSFSAQVIVLNHPGTIK AGYSPVVDCHTAHIACKFESLDTRIDKRTGKTLEE NPKTIKNGDAAMVTMKPNKPMVVETFTDYAPLG RFAVRDMRQTVAVGIIKAVEKKDPSSAKVTKSAV KAGKK BmR1_04g05965 MTKIISACGREVLDSRGNPTVECEVTTEGGKFRAI 49 VPSGASTGIYEALELRDGDKTRYLGKGVQNAIKN MHNIICPGIQGFLCTEQEKLDNHMVKVLDGTQNE WGFSKSKLGANTILAVSMGAARAGAAAKGIPLY EHLAQLSGKPTDKFIMPVPCLNVINGGSHAGNAL AFQEFMILPTVADNFSNALRMGVEVYHTLKKVIN KKYGQDATNVGDEGGFAPNISTPQEALDLLVEAI AAAGYTGKIKIAMDVAASEFYQKDVKMYNLTFK SSSPDIKTSDQLVELYKELVNKYPIVSIEDPFDQDD WEAYAKLTAAIGDKIQIVGDDLLVTNPKRIEAAIQ KKACNALLLKVNQIGSVTESIQACKISQENGWGV MVSHRSGETEDVFISDLVVALGTGQIKTGAPCRSE RNAKYNQLLRIEQELGERATYSKVFNK BMR1_03g01010 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGI 50 PPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVL RLRGGD BMR1_03g01340 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGI 51 PPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVL RLRGGMQIFVKTLTGKTITLEVEPSDTIENVKAKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKES TLHLVLRLRGGMQIFVKTLTGKTITLEVEPSDTIE NVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDY NIQKESTLHLVLRLRGGVIDPSLALLAQKYNCNK MVCRKCYARLPPRATNCRKKRCGHCNDLRPKKK IKGN BMR1_03g02390 MGDDDNAALVVDNGSGNVKAGIAGDDAPRCVF 52 PSIVGRPKNPALMIGMDEKEVYVGDEAQSKRGIL TLKYPIEHGIVTNWDDMEKIWHHTFYNELRVNPE EHSVLLTEAPLNPKTNREKMATIMFETHNVPAMY VAIQAVLSLYSSGRTTGIVLDSGDGVSHTVPIYEG YAMPSAIMRLDLAGRDLTEYMQKILVERGFSFTT SAEKEIVRDIKEKLCYIALDFDEEMQAAETSSDLE KSYELPDGNIITVGNERFRCAEVLFQPSFIGKECH GLHKTTFDSIIKCDVDIRRDLYSNVVLSGGTTMLQ GIGERLTKELSCLAPSTMKIKVVAPPERKYSVWIG GSILSSLSTFQQMWITKDEFDESGPVIVHRKCF BMR1_02g03540 MHNHSYYTILLTIALIHTTGCHGFLHRNIKFILHSM 53 TVYGKPKRLHKTPPWAHLFEEKVEPSPLGEPWAK LSKDVANGERRVRLTVKKSHLQIYAAVVDDYKN QVICIASSNLPVLADVLGTVPTKDPTVRRNKGNN VKAAYEVGKHIGRLALAKGVAKVYFDRAGYKY HGRVEAVAIGARKVGLQL BmR1_04g09800 MYQIDRMIDKHKDPIDPLQTILSVKGTMKCKLSE 54 MLTRHSTEDRHSLSLVADLKRESPTHTDSRSGVR LSFLDAGEVVVTMANTGFDVVLVNTDDIAYKGT LDDLKTSICAAHAIGNRSRPAVVMKDIILHPLQLA QAAQLHADGVVLNSFYLGPALESMIDTSYNLGIE PIVEVHTLEDALYAIQLHTKILMINQWDRLTNKC HPNRALQIREIVPDGIITIACGGIKTLEQIEQLGLAG YDAVVLGKKLADTNIPSFVGSIKKWNAPGKGILAI SKPLFFTDEMDEKDVSGGGHVIRMKQNYRQTLQ EMCEFYQIAENNCVEAPRPKDVLLRFIGNDMPEK WIFKREKWMKDHAHEYPSEDHATAVYDFNLAVE LNTTLECNKKLLSQHFKREMVDKLEEAIKNYVKK CYINLKRFTNKISCS
TABLE-US-00007 TABLE 7 B. microti secreted antigens identified in the hemolysate fraction: SEQ ID NOs: 55- 62 Antigen ID Annotated Sequence SEQ ID NO: BMR1_01g02545 MSQGPAIGIDLGTTYSCVGVWKNETVEIIANDQG 55 NRTTPSYVAFTDVERLVGDAAKNQDARNPENTV FDAKRLIGRKINDPCIQSDIKHWPFTVAAGPNDKP VIKVQFQGETKSFHPEEISSMVLTKMKEIAESYLG KTISNAVITVPAYFNDSQRQATKDAGTIAGLNVM RIINEPTAAAIAYGMDKKGTSEKNVLIFDLGGGTF DVSILTIEDGIFEVKATQGDTHLGGEDFDNRLVNF CVDDFKRKNGGKNISTNRRALRRLRTQCERAKRT LSHSTQATIVVEAIFDGIDYSCNITRARFEELCAEM FKNTLIPVEKALADADMDKKQIHEVVLVGGSTRI PKIQQLIKDFFNGKEPCKSINPDEAVAYGAAVQA AILTGEQSSKVQDLLLLDVTPLSLGLETAGGVMT VLIPRNTTIPAKKEQEFTTNENNQTGVMIQVFEGE RSMTCDNNLLGKFHLTGIPPAPRGVPQIKVTFDID ANGILTVSAADKSTGKTEHVTITNDKGRLSQQDID RMVAEAEKFREDDEKKKRCVESKNELENYCYSM KNALEEEGVKSKLSSSELSEAQKLLQNTFSWIESN QLAEKEEFEAKLKEVQAVCTPLTAKLYQAGGGV PGGAAPGGFNAGGAAPSGPTVEEVD BMR1_02g03395 MTAYIPGLLERLSAPIVSSRLDDDDLAYLGKYESE 56 ISDSDNLQNIYSNDTIVALGDLDAKYKQQTYLSSL NSHIVDQDAQIAFYPVKYTNKIIRNDSNLTYRRTT SSLSNPLVNYIQKSQHDTPDPVDSNIQNSSSEQYAI TTASSGSDKSKRLSHDNSKTDHDIHDKSGVVTAN NHSTDRFQNAGDLVWQPGFTRQLGTKGRRVLLD LIRKVYRGNPEYFKNILALKNPPTSISNLPFCNVT MLWELANDFGVFDQAIQIHYAHGKPGYPANYHN TANYGCNKTRKTNSGKSKKNKFYNYDYEAYDEY YEDEYISSFSNGRTSRGRKIVQPKRFDDYDHLLDS NYQVEYGYCSNRHLNKDGNTILYYKNKSHDYVN LGELKGHKIERFTSQLIC BMR1_03g04775 MKNDFNSVELPGHFLFTSESVNEGHPDKLCDQIS 57 DSILDACLEQDPESKVACEVCTKRGMVMVFGEIS TNAKVNYEEVVRNVVKNVGYDSEDKGIDYKTM DVIINLDQQSHEIAQAVHLGKDADNTCAGDQGIM FGYATDETPEYMPLSHSLATGLGKRLKDVRLSGL LPYLGPDGKTQITVEYIKEGYGSIKPIRVHTVLISQ QHSANVSNDKLREDLMTHVVKAVIPPHFLDDKT KYYLNPSGHFVVGGPSSDAGLTGRKVIVDTYGG WGAHGGGCFSGKDGTKVDRSAAYYARKVAKSL VANGFCRRALVQVSYSIGIRSPLSLHVDSYNTCIE GFTDLDLEQIAVRNFDFSVGNIIKELQLKKPIYSQT GVYGHFGKDNPEYLWENVKDLSHELTHKPKR BMR1_03g03380 MDYTRSLFTLSGPATASEVEKHIQNAIEFVKRRDP 58 DQVQFIQAFTEVANGLAPVFQTDLKYLEIFLSLSE PERVITFKVPWVNDAGKLMINRGFRVQFNSTLGP YKGGLRFHPSVNLSILKFLGFEQIFKNSLTTLAMG GGKGGSDFDPKGKSDNEVRSFCQSFMTELQRHIG PDTDVPAGDIGVGEREIGFMYGQYKRLSNSSTGT LTGKDPKWGGSFIRPQATGYGLVFFVQYILNDLH NGDSFKGKRVAISGSGNVAQYAADKVIDFGGIPIT FSDSSGYIYEPNGFTKEMVTVLMELKNIQRARVS EFLKYSNTAKFFPNKKAWDVDTNVNVALPCACE NELDKADAEMLVKKGCIIVGEGANMPTTPEAISV FKAAKVTVCPGKAANAGGVAVSGLEMSQNSQRE KWTSEKVLEKLQDIMKNMSKACQEAAAKYNVH GDIISGANIAGFLKVAHSYCDQGCV BmR1_04g08050 MSYSAEETDASIEQWKILRLIRNLESAKGNGTSMI 59 SLIIKPKDEIARINKMLADEFGTASNIKSRVNRLSV LSAITSTQQKLKLYRQTPPKGLVVYCGTILTEDGK EKKVSLDFEPFKPINTSLYLCDNKFHVEALKELLE SDEKFGFIIVDGNGVLYGTLQGNTKEVLHSFTVDL PKKHGRGGQSALRFARLRLEKRHNYVRKVADIA VQMFITNDRPNVSGLVLAGSADFKNDLMSSDMF DPRLAAKVVKIVDVSYGGDHGFNQAIELSAQCLS NVKFIQEKKIISRFFDELAHDTGRYVYGVHDTINA LEMGAVEMLIVYEALDIQRLQMRNPVTGEESVIIQ TSERDTEAMRDPVNNVDLELVESIHLSEWLVNNY RNYGATLEFITNKSQEGSQFHRGFGGIGGILRYKL DMSEYDLPVNDNDFDDFI BMR1_02g01430 MDSLVPPYNKLNFDISRPDISGNTLHYTFFQYPDS 60 SISKLRNVFAHNPQQNFTNQPFYPPKHNDTPHTEQ NGSQFIHSNSNTSNNLESNDVDNNASASACDKRS FPHDDRYSSSNYNEYPGILSSIQDIADLFDLDNYHI YHGIDNISYLTYSTDANRTGIDSKLEFIYEVLNSNG SNMTLSKLEGFLRILTTDDNPIGTLTPYGSLMAHT CLRFLKSYIRTDNKGNSHDPDTFIYSIEGKKGKSK LSKQGITNENETTTVSSDFRRIAMFNFELYSKQLM DTIHSQDKSIAINKRKFDDLTSNYTKSESVSIDSMS AISDSTDKRAVSSQTLKIIKQGSAYLQDFISKYRIG FEPHFPRIVCGSIDRSLVTNQASITVPIDKSGRSVHI AFEALSDSYGMSVWPELNQLPNSLQLGTKIQIAW HKTPGNMRWYIGTIVQSTASQYSVQVYLGKNEY KTRYCTMKYFVPPNVPVVKPDNTWWRLVPREIN LSTDLYVGSCVSLYDIISNFESIDCVICNVYYSNDN SPNGPIKGPNEEIYKSDKSSSGFKIQLNCACHGPTR RIRQGVLTTGVVRVHIYCMSHKIDKIIPIDSIRGYK LNYDLHAHFDGQPDYGPLMQSTTWTWVIPRLVI APDNKYDDEKYWTSNPKYSLLIFPYHRNMEYIIS ATKLIAILHPELNVTSLYNNGEWFVRQEGDIAIKG YGGLRPSGLSFSKIPLLDRLFGCHRIKVFYNVPIDV NKKVEDFKNDYTISRCANCRGIYYTDISHASNGK TYISSDVEYRKKIQIAAAIRWKRVRESSNTILNCY NENDELVYRTKNSFSLSWEQDGRNLELAYRRAH APGISKFESKRLDSLMKSNKQVSNVPENMCTEGL LEKFNESMKFSESVKSTDSKNAAKLLEVAILGGW MNDSTKTESINTNKGDNIFRYINKSIQAKAAQDAL DFLKNCSPPCLSKQVGKHYTPINGKQSMFKQGG MSDYQDSIPHLDIGNPMISDYNSKKVKLQSVSGD QRGLNIYENPCIATVHDPSVNYNSLRNFLAKAEW DEEIEDAPSHGVLDFTIKSEEPVHNEAGEDCIYAV PINDSVNIIWSDTEDAK BmR1_04g08520 MVERLIARSLSIDSIETQILRFFSGDIGLQTVLLEFQ 61 KGPRAFVICLDIINKHCKEFSSKSLPLILFCSQTLVE CDHIYSLCRLVSDGQAQDLDKSDATKLDTEREETI EKLERVINLLEQLNKVNPQIFPSLRFLLCAWLRLK LFDNWSKGITFEIIQFVGTFQSNRQFQIELLAILAQ EICNDKFILSIARRNELAKNCISQAPMVFKFLGVK DSSIGSVYSDWIQLHVKYLDSIVDEEQCELPSDVD LMVKLSLSTLISLNLIDKLFEQSPLPIHTITNLIVLC PLDNHTALQDDCDMINPLVNEILTLIICNLKLYKI QSPLFKTTSPILLSLSYEQLPYLIEHQFSEDIDNILD GTIRILSNGDYPTRDVMVNFWINLKRNINTQNGW EKLADYLQKIVIVFYEMPLYETEKYDFAELCSFRE SASMLLFEIADAIGHQFIFDIVECRLQVLVETINLK EQITVSFSEIESIFFILSAISSNAEMGKDTCIPTALAL LNKLKYPTQGSLSLLLSISIGRLILWTAEYSGKKTD LFNCLFMLIIGTLLPSILRIKNNSASKGMYYYLLGE NILIDAMLALSRTSRKIVSEDTQEVKKYLICIYQIIK NVQFSIENRIKAVNAAGAIISYMPIAEMKTLFSDLI ENLSNNIKSVSNPTDYIQLYLMAMQSVCPIPDCIE SEVAILKIVEKHVSVMELLFTSSNEDIIERLSQVLV VVNRISRNHSEASPLFLWTLKLLSVSFRCSHPSHP YSALRSILINVNDCSVENWASISNSLLPSLAMLKD SIVSCCVGDKDRNLLQQQMSDLTMDFNTSQLLSA PDSVGLCVDCLNVALNRHEIANFLLDKSKFIIFLES LLIILPYIVHPKVLHACMILIKLVASIDMNEASGND YSDSAVIGTSVYAKKIRLLNIISQRCNESSDEIHTIT FKIVTAILSTVISGTCGVETWIDVAAETILALMCD GKTADEAKNAIDSFFEMISEPSINVTIKNDYCNKL KVPNTLFQALLQLEGWKLWKKC BmR1_03g00947, MRGMFSNKWMSFVCFSILFVALKSDLEYVSALKL 62 BmIPA48 LRAPPQTSLFLEKLIDDGSDIPKDPIDTDKEESQSS LFKFNLNLFN KKSIWEADEKFVITLAKSRLNVILAQKLDKFLAKT CKIYTVDSEHSACINDIKIYAQKCIESNDLNSCYVI PIQPIAKLP TSRLYGLVPHVLNFSILIFTNLRSNLDRYYIDGSKD WFSHIFMRLKRFFGIRNKHSYFSDNRLMNKIFSRT STTFGPDRS DSLLSNYIKFGAIEYAILLNTRSNLVKMILSSFAHI KFVRKRLYKFYTNKWKSIEGLVTRGHLKPVDLSN NPISDNIFKY FGKFSNNTNLSNAIAGAFLDHYKSLFSNSTDVNGE GSSGEGPSGEGFNGEGSSGEGPSGEGFNGEGFDG EGPSGEGPSGE GFNGEGFNGEGLNGEGPSGEGPSGEGLNEWNGL MNGTA
EQUIVALENTS
[0092] Although preferred embodiments of the invention have been described using specific terms, such description is for illustrative purposes only, and it is to be understood that changes and variations may be made without departing from the spirit or scope of the following claims.
INCORPORATION BY REFERENCE
[0093] The entire contents of all patents, published patent applications, and other references cited herein are hereby expressly incorporated herein in their entireties by reference.