Ehrlichia immunodominant proteins

Abstract

Ehrlichia spp. are tick-transmitted, obligate intracellular bacteria that cause disease in animals and humans, ranging from mild to severe and life-threatening. Due to the potential severity of ehrlichiosis, clearly there is a need for proteins that can be used for diagnosis of or vaccination against Ehrlichia. Methods and compositions for diagnosing and vaccinating against Ehrlichia canis and Ehrlichia chaffeensis are provided.

Description

EHRLICHIA IMMUNODOMINANT PROTEINSBACKGROUND

[0001] This application claims priority to provisional Application No. 63 / 611,337 filed December 18, 2023, entitled “Ehrlichia Immunodominant Proteins”, the entire contents of which is incorporated herein by reference.

[0002] This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said XML Sequence Listing, created on December 13, 2024, is named CLFRP0545WO.xml and is 11,144 bytes in size.1. Field

[0003] The present disclosure relates generally to the field of molecular biology and medicine. More particularly, it concerns polypeptides and methods that can be used for diagnosing, treating, and vaccinating against infection by Ehrlichia.2. Description of Related Art

[0004] Ehrlichia spp. are tick-transmitted, obligate intracellular bacteria that cause disease in animals and humans, ranging from mild to severe and life-threatening (Paddock and Childs, 2003; McBride and Walker, 2011). E. chaffeensis is the etiologic agent of human monocytotropic ehrlichiosis (HME), an emerging zoonosis. E. canis is the etiologic agent of canine monocytic ehrlichiosis (CME), a highly prevalent globally distributed hemorrhagic disease in dogs (Paddock and Childs, 2003; Harrus and Waner, 2011).

[0005] Conventional immunoblotting approaches have helped identify major antibody reactive proteins from E. chaffeensis and E. canis, including major outer membrane proteins (OMPs) (Ohashi et al., 1998a; Ohashi et al., 1998b), tandem repeat proteins (TRPs) (Doyle et al., 2006; McBride et al., 2007; Luo et al., 2008; Luo et al., 2009; McBride et al., 2011) and ankyrin repeat proteins (Anks) (Nethery et al., 2007; Luo et al., 2010), that contain linear antibody epitopes. Many of these proteins elicit protective immune responses in Ehrlichia infection models (Li et al., 2001; Crocquet- Valdes et al., 2011; Kuriakose et al., 2012; Nambooppha et al., 2022). Moreover, experimental studies have demonstrated protection against infection using live attenuated vaccines and subunit vaccines (Rudoler et al., 2012; McGill et al., 2016; Budachetri etal., 2022); however, there are no commercial human or veterinary vaccines available for HME or CME.

[0006] Until recently, the number of defined E. chaffeensis and E. canis antigenic proteins has been limited (McBride and Walker, 2010), compared to a large number of antigenic proteins (~7-20% of the proteome) identified in other pathogens, such as Chlamydia, Coxiella, Burkholderia and Bartonella (Barbour et al., 2008; Feigner et al., 2009; Vigil et al., 2010a; Vigil et al. , 2010b; Cruz-Fisher et al. , 2011 ; Vigil et al. , 2011). Since the completion of whole-genome sequencing of the first bacterium Haemophilus influenzae in 1995, a variety of multiomics approaches are now available, including genomics, proteomics, transcriptomics, and immunomics which integrates these omics approaches to study the immunome (Sette et al., 2005; Loman and Fallen, 2015; De Sousa and Doolan, 2016; Babu and Snyder, 2023). The immunome is defined as the set of antigens or epitopes that interface with the host immune system. Genome-based in silico reverse vaccinology has also accelerated the identification of new vaccine candidates, but it largely relies on the accuracy of a serial of computational prediction tools, which are limited by the validated protective antigens in which the algorithm is based (Bidmos et al., 2018). Due to the potential severity of ehrlichiosis, clearly there is a need for proteins that can be used for diagnosis of or vaccination against Ehrlichia.SUMMARY

[0008] The present disclosure overcomes limitations in the prior ait by providing new immunodominant proteins that can be used to diagnose or vaccinate against Ehrlichia. As shown in the below Examples, immunodominant proteins (having an optical density using ELISA OD650 of greater than 1) comprise about only 3% of the proteins produced by Ehrlichia (e.g., FIG. 5). Significant differences and unpredictability can exist between in silica prediction software and actual immunological data observed in vivo. Immunomics-based strategies are used to test approximately half the E. chaffeensis and E. canis proteomes, and an overview of the screening strategy utilized that involved both in silica and experimental methods is shown in FIG. 6. A variety of antigenic proteins from E. chaffeensis and E. canis were identified (see FIGS. 1A-C), and from these proteins, only a select few of these proteins were experimentally determined to be immunodominant by testing for ELISA reactivity with serum samples from humans or dogs that had been previously infected with E. chaffeensis and E. canis, respectively. As shown in FIGS. 2A-B, Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NO:2), Ech_0350 (SEQ ID NOG), Ech_0506 (SEQ ID NOG), Ech_0679 (SEQ ID NOG), Ech_0494 (SEQ ID NO:6), and Ecaj_0647 (SEQ ID NOG) and Ech_0905 (SEQ ID NOG) surprisingly displayed immunodominant responses against E. chaffeensis having an optical density using ELISA OD650 of greater than 1 in sera samples from humans previously infected with E. chaffeensis, and Ecaj_0647 (SEQ ID NO:7) similarly displayed immunodominant responses against E. canis in sera samples from dogs previously infected with E. canis. All of these proteins also surprisingly resulted in at least strong immunoreactivity (ELISA OD650 > 0.5) with all sera samples tested. Data provided herein shows that these proteins can be produced using in vitro transcription and translation (IVTT) to produce immunoreactive and immunodominant proteins. It is anticipated that these proteins can similarly be used to produce immunological responses against Ehrlichia, e.g., when administered (e.g., intramuscularly, intranasally, etc.) in a pharmaceutical or vaccine composition. Denaturation of the proteins showed conformation-dependent immunoreactivity of all of the above proteins (FIGS. 3A-C), except for Ecaj_0647 (FIGS. 4A-B). It is anticipated that a polypeptide having at least 90%, more preferably at least 91, 92, 93, 94, 95, 96, 97, 98, or at least 99% sequence identity to any one of Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NOG), Ech_0350 (SEQ ID NOG), Ech_0506 (SEQ ID NOG), Ech_0679 (SEQ ID NOG), Ech_0494 (SEQ ID NO:6), and Ecaj_0647(SEQ ID N0:7), or Ech_0905 (SEQ ID NO:8) can be used to diagnose exposure to ehrlichiosis or stimulate an immune response against E. chaffeensis or E. canis.

[0009] As described in the below Examples, the remaining portion (-50%) of the E. chaffeensis and E. canis proteomes (n - 444 and n - 405 proteins, respectively) that have not been previously examined were analyzed in order to identify the complete immunomes of these important pathogens. Almost half of the E. chaffeensis proteins screened (196 / 444) reacted with antibodies in convalescent HME patient sera, while only 43 E. canis proteins reacted with CME dog sera. New major immunoreactive proteins were identified in E. chaffeensis (n = 7) and E. canis (n = 1), increasing the total number of E. chaffeensis (n=14) and E. canis proteins (n=18) that exhibited antibody reactivity comparable to well-defined major antigenic proteins (TRP120 and TRP19). All of the E. chaffeensis but only some E. canis major immunoreactive proteins contained major conformation-dependent antibody epitopes. The E. chaffeensis immunoreactive proteins were generally small (< 250 amino acids; ~27kDa) and the E. canis proteins were slightly larger (> 320 amino acids; -35 kDa). The majority of these new Ehrlichia major immunoreactive proteins were predicted to be type I secreted effectors, some of which contained transmembrane domains. Immunodominant proteins Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NO:2), Ech_0350 (SEQ ID NO:3), Ech_0506 (SEQ ID NO:4), Ech_0679 (SEQ ID NO:5), Ech_0494 (SEQ ID NO:6), Ech_0905 (SEQ ID NO:8), and / or Ecaj_0647 (SEQ ID NO:7)) can be included in a subunit vaccine for the ehrlichioses.Table 1. Immunodominant ProteinsEch_1061 : MKIIYGYPNQKGNVNS VLAFGNFDGIHLGHQSIINAIKNIS VREKITS VV ITFSPHPAEYLRNKQNFLLLDLEQKIELLQSYGIDYLYIIDFNESFAQLS PDAFIKDVLVNSCRIKYIVVGHSCFFGHKCLGNIDLLYSYANIYDYEIVR IDPIFINDKLCSSSLIREYLSEGKVDLASKILGRPYQINGKVIKGLARGR VIGFPTVNVDIKHILVPRVGVYSACIKIDDNNVWLNGIVNIGFRPTFNDL SFPILEMHIFDFDSDIYNQHVAIQLLDFIRPERKFSSIEQLKQQINEDII QVKKSLKYIGSKDDEVV (SEQ ID NO: 1);Ech 0725 : MLIFDRALVRFYRDRLSCNKDNDFIFS AISDILLDKI ALFS VATGLALNLGVRTNSFVENLLHKKLISSKEQVVQCDLSYCVLYNVNGGYKIVADEEALPFRNNVFDLVISNVSLHNVNNLFSVLLNIYNIIKSKGVFLAALFGSKTLYELKHSIIRAEMDFGIAPRVLPFINVQDIISLLQKIRYSDIVVDVNTIVVKYSDIYTLFRDLKNMGEGNVLRVRNKYPLTRTVITKIFENYKQYFSVDKISI PATFEIITLKGSKV (SEQ ID N0:2);Ech 0350: MFNDVVMLALGSNCGNMLAYIKSAINMLPLHNKNYSYLYKTPALLPENAADHWDTPYLNMVVSGYTNLSSSAMLERIKSIEKMLGRINHQRWAPRPIDIDIILWGNKVVDSQTLSIPHQQMHCRDFVLVPLCDIYARFIHPVLQIPIYEM LLSLNNINLIKQNSHVLQQ (SEQ ID NOG);Ech_0506: MLQFKILSCKAIVLLLSMYSILCFQYLNFYRISQHNKIYTSLLNNVFYSS KLLFEYFYTILNTLFITSKHTIITKSTISHYKSHINLNITDFQRLN (SEQ ID NO:4);Ech_0679: MKAILYLQIILLVLICLTHSKVFAKSMVSDSVSKFYVGGSYGYDKKILEFKTGDGYLDFDFNVNNVTGYNAGLTLGYICPSASLNNFRTDLEFMYITKKKIDNNKEVVDEDREKVLQKKMLYWNNNKYKVFFNLYYDVGNFFSIRDFTFFFGAGVDLHNF1GKLLKNTETLDLKSNLMMQS1VG1KYQLLNN11MYGGYR YFIGYMKPKDNINFKRCYILGSYGLMFGLEFVF (SEQ ID NOG);Ech_0494: MAGTVKADQLFKGLTRPTMLFGVSYIFAMLNFMICIMIFMYTNDFRALFI LGPGIHSVGFLASSKEPLFLELFMLRMKKCSKCLNRFYHNANSYDVM (SEQ ID NO:6);Ech_0905: MQLYTIVATWFGCGNITRAPGTIASFATMLLSPAIILNNLFGILIIVLTGIMGLFAIPKYLLDHPNIVDPKEIVIDEVIGQLIAFSIPIVFFRYYQYVPQTFNTLYLLFYIKILITSFILFRIFDITKIWPINILERISGTTGIILDDVL AGIMSSICTIFIIAKIGT (SEQ ID NOG);Ecaj_0647: MANVVVTGEQLDKAIREVVHILEDAVGCTAGPKGLTVAISKPYGAPEITKDGYKVIKSIKPEDPLALAIANIIAQSASQCNDKVGDGTTTCSILTAKVIEEVSKAKAAGADIVCIKDGVLKAKEAVLDALMSMKREVLSEEEIAQVATISANGDKNIGVKIAQCVQEVGKDGVITVEESKGFKELDVEKTDGMQFDRGYLSPYFVTNSEKMLVEFENPYILLTEKKLNIIQPILPILENVARSGRPLLIIAEDVEGEALSTLVLNKLRGGLHVAAVKAPGFGDRRKDMLGDIAILTGAKHVISDDLAIKMEDLTLAELGTAKNIRITKDTTTIIGSVDNSSDNVQSRINQ IKVQIESSTSDYDKEKLRERLAKLSGGVAVLKVGGSSEVEVKERKDRVED ALHATRAAVEEGVVPGGGAALLYTLSVLENLKSKNDDEQLGINIIKRALQ APIKRIIRNSGSENAPCVIAHLLKQNDKELIFNVDTMNFANAFTSGVIDP LKVVRIAFDFAVSLAAVFMTLNAIVVDVPSKDDAGAAGGAGGMGGMGGMG GF (SEQ ID N0:7)

[0010] An aspect of the present disclosure relates to pharmaceutical composition comprising a nucleic acid comprising an open reading frame encoding Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NO:2), Ech_0350 (SEQ ID NO:3), Ech_0506 (SEQ ID NO:4), Ech_0679 (SEQ ID NO:5), Ech_0494 (SEQ ID NO:6), Ech_0905 (SEQ ID NO:8), Ecaj_0647 (SEQ ID NO:7), or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto; wherein the composition is formulated in a lipid nanoparticle or a viral vector. The nucleic acid may be a ribonucleic acid (RNA). The nucleic acid may be an mRNA further comprising a 5' untranslated region (UTR) and a 3' UTR. The mRNA may comprise at least one analogue of a naturally occurring nucleotide or wherein the mRNA is chemically modified. The analogue may be selected from the group consisting of phosphorothioates, phosphoramidates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, and inosine. The mRNA may comprise pseudouridine, a 5' cap analog, or a poly(A) tail. The 5' cap analog may be 7mG(5')ppp(5')NlmpNp. The chemical modification may be a 1 -methylpseudouridine modification or a 1 -ethylpseudouridine modification. The mRNA may comprise a 5' untranslated region (UTR) and a 3' UTR. The mRNA may be comprised in liposomes, lipid nanoparticles, or a viral vector. The liposomes or lipid nanoparticles may comprise an ionizable cationic lipid, a neutral lipid (e.g., DSPC), sterol (e.g., cholesterol), and / or a PEG-modified lipid (e.g., PEG-DMG or PEG-DMA). The RNA may encode the polypeptide for secretion. The RNA may encodes the polypeptide as an intracellular protein. The polypeptide may be comprised in a fusion protein, preferably wherein the fusion protein comprises a transmembrane region. The nucleic acid may be a DNA. The DNA may be comprised in a viral vector. The viral vector may be an adenovirus, or an adeno-associated virus (AAV). The pharmaceutical composition may be further characterized as an immunogenic composition, an immunoreactive composition, and / or a vaccine composition.

[0011] Another aspect of the present disclosure relates to a pharmaceutical composition comprising Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NOG), Ech_0350 (SEQ ID NOG), Ech_0506 (SEQ ID NOG), Ech_0679 (SEQ ID NOG), Ech_0494 (SEQ ID NOG), Ech_0905 (SEQ ID NO:8), Ecaj_0647 (SEQ ID NOG), or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, and an excipient. The Ech_1061, Ech_0725, Ech_0350, Ech 0506, Ech_0679, Ech_0494, Ech_0905, Ecaj_0647, or the polypeptide having sequence identity thereto may be recombinantly expressed by a cell, and purified or substantially isolated, before inclusion in the pharmaceutical composition. The composition may further comprise an adjuvant. The adjuvant may comprise a triterpenoid saponin (preferably Quil A), a sterol, and / or an immunostimulatory oligonucleotide (preferably a CpG-containing ODN). The CpG-containing ODN may be 5’ JU*C-G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C* G*T 3' (SEQ ID NO: 9), whereinrefers to a phosphoro thioate bond,refers to a phosphodiester bond, and “JU” refers to 5'-Iodo- 2'-deoxyuridine. The composition may comprise an E. canis bacterin or an E. chaffeensis bacterin; preferably wherein the E. canis bacterin or the E. chaffeensis bacterin is a heat-inactivated or chemically-inactivated bacterin, preferably wherein the chemically-inactivated bacterin was inactivated with formaldehyde, formalin, bi-ethylene amine, radiation, ultraviolet light, betapropiolactone treatment, or formaldehyde. The Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NOG), Ech_0350 (SEQ ID NOG), Ech_0506 (SEQ ID NOG), Ech_0679 (SEQ ID NOG), Ech_0494 (SEQ ID NOG), Ech_0905 (SEQ ID NOG), Ecaj_0647 (SEQ ID NOG), or the polypeptide may be comprised in a multimer or fusion protein. The pharmaceutical composition may be further characterized as an immunogenic composition, an immunoreactive composition, and / or a vaccine composition.

[0012] Yet another aspect of the present disclosure relates to a method of detecting antibodies that specifically bind an Ehrlichia organism in a test sample, comprising: (a) contacting a polypeptide comprising or consisting of a sequence of Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NOG), Ech_0350 (SEQ ID NOG), Ech_0506 (SEQ ID NOG), Ech_0679 (SEQ ID NOG), Ech_0494 (SEQ ID NOG), Ech_0905 (SEQ ID NOG), Ecaj_0647 (SEQ ID NOG), or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, 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 anindication 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 organism 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 multiplex immunoassay, a mass spectrometry assay, or a particulatebased assay. The step of detecting may comprise a lateral flow assay or an enzyme-linked immunoassay, wherein the enzyme-linked immunoassay is an ELISA. The polypeptide may be further characterized as an isolated polypeptide.

[0013] Another aspect of the present disclosure relates to a method of identifying an Ehrlichia infection in a mammalian subject comprising:(a) contacting a biological sample from the subject with an polypeptide comprising or consisting of Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NO:2), Ech_0350 (SEQ ID NO:3), Ech_0506 (SEQ ID NO:4), Ech_0679 (SEQ ID NO:5), Ech_0494 (SEQ ID NO:6), Ech_0905 (SEQ ID NO: 8), Ecaj_0647 (SEQ ID NO:7), or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, 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 multiplex immunoassay, a dipstick test, or a particulate-based assay. The subject may be a human or a dog. The polypeptide may be further characterized as an isolated polypeptide.

[0014] Yet another aspect of the present disclosure relates to a polypeptide comprising or consisting of Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NO:2), Ech_0350 (SEQ ID NO:3), Ech_0506 (SEQ ID NO:4), Ech_0679 (SEQ ID NO:5), Ech_0494 (SEQ ID NO:6), and Ecaj_0647 (SEQ ID NO:7), Ech_0905 (SEQ ID NO:8), Ecaj_0647 (SEQ ID NO:7), or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, wherein the polypeptide is immobilized on a surface of a support substrate. The support substrate may comprise latex, polystyrene, nylon, nitrocellulose, cellulose, silica, agarose, or magnetic resin. The supportsubstrate may be a reaction chamber, a well, a membrane, a filter, a paper, an emulsion, a bead, a microbcad, 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 polypeptide may be comprised in a kit. The polypeptide may be produced via peptide synthesis or in vitro transcription and translation (IVTT). The polypeptide may be recombinantly produced. The polypeptide may be further characterized as an isolated polypeptide.

[0015] Another aspect of the present disclosure relates to a polypeptide comprising or consisting of Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NO:2), Ech_0350 (SEQ ID NO:3), Ech_0506 (SEQ ID NO:4), Ech_0679 (SEQ ID NO:5), Ech_0494 (SEQ ID NO:6), Ech_0905 (SEQ ID NO:8), Ecaj_0647 (SEQ ID NO:7), or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, wherein the polypeptide is covalently attached to or bound to a detectable label. The detectable label may be a fluorescent label, a radioactive label, an enzyme label, or a luminescent nanoparticle. The luminescent nanoparticle may be a luminescent rare earth nanoparticle, a luminous nanoparticle, or a strontium aluminate nanoparticle. The polypeptide may be comprised in a kit. The polypeptide may be produced via peptide synthesis or in vitro transcription and translation (IVTT). The polypeptide may be recombinantly produced. The polypeptide may be further characterized as an isolated polypeptide.

[0016] Yet another aspect of the present disclosure relates to a kit comprising: (a) the polypeptide described above or herein, (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 polypeptide may be immobilized on a membrane or a microtiter plate. The reporter molecule may be selected from the group consisting of luciferase, horseradish peroxidase, a luminous nanoparticle, P-galactosidase, and a fluorescent label. The luminous nanoparticle may be a strontium aluminate nanoparticle. The kit may further comprise a dilution buffer for dog or human serum. The kit may comprise a lateral flow immunoassay or a lateral flow immunochromatographic assay. The kit may comprise an enzyme-linked immunosorbent assay (ELISA).

[0017] Another aspect of the present disclosure relates to a method of inducing an immune response in a mammalian subject comprising administering to the subject an effective amount ofa pharmaceutical composition comprising a polypeptide comprising or consisting of Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NOG), Ech_0350 (SEQ ID NOG), Ech_0506 (SEQ ID NOG), Ech_0679 (SEQ ID NOG), Ech_0494 (SEQ ID NOG), and Ecaj_0647 (SEQ ID NOG), Ech_0905 (SEQ ID NOG), Ecaj_0647 (SEQ ID NOG), or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, or a nucleic acid encoding a polypeptide sequence of Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NOG), Ech_0350 (SEQ ID NOG), Ech_0506 (SEQ ID NOG), Ech_0679 (SEQ ID NOG), Ech_0494 (SEQ ID NOG), Ech_0905 (SEQ ID NO:8), Ecaj_0647 (SEQ ID NOG), or encoding a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto. The nucleic acid may be an mRNA. The mRNA may comprise at least one analogue of a naturally occurring nucleotide or wherein the mRNA is chemically modified. The analogue may be selected from the group consisting of phosphorothioates, phosphoramidates, peptide nucleotides, methylphosphonates, 7- deazaguanosine, 5-methylcytosine, and inosine. The mRNA may comprise pseudouridine, a 5' cap analog, or a poly(A) tail. The chemical modification may be a 1 -methylpseudouridine modification or a 1 -ethylpseudouridine modification. The mRNA may comprise a 5' untranslated region (UTR) and a 3' UTR. The mRNA may be comprised in liposomes, lipid nanoparticles, preferably wherein the liposomes or lipid nanoparticles comprise an ionizable cationic lipid, a neutral lipid (e.g., DSPC), sterol (e.g., cholesterol), and / or a PEG-modified lipid (e.g., PEG-DMG or PEG-DMA). The nucleic acid may be a DNA. The DNA may be comprised in a viral vector (e.g., an adenovirus, or an adeno-associated virus (AAV)). The method may comprise administering the pharmaceutical composition described above or herein to the mammalian subject, preferably a human or a dog. The pharmaceutical composition may be administered subcutaneously, intramuscularly, nasally, via inhalation or aerosol delivery, or intradermally. The polypeptide may comprise or consist of Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NOG), Ech_0350 (SEQ ID NOG), Ech 0506 (SEQ ID NOG), Ech 0679 (SEQ ID NOG), Ech_0494 (SEQ ID NOG), Ecaj_0647 (SEQ ID NOG), Ech_0905 (SEQ ID NOG), or Ecaj_0647 (SEQ ID NOG). The method may further comprise administering an Ehrlichia bacterin or an adjuvant to the mammalian subject. The pharmaceutical composition may be further characterized as an immunogenic composition, an immunoreactive composition, and / or a vaccine composition.

[0018] Yet another aspect of the present disclosure relates to a method of treating an Ehrlichia chaffeensis infection in a subject comprising: (a) contacting a biological sample fromthe subject with an isolated polypeptide comprising or consisting of a sequence of Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NO:2), Ech_0350 (SEQ ID NO:3), Ech_0506 (SEQ ID NO:4), Ech_0679 (SEQ ID NO:5), Ech_0494 (SEQ ID NO:6), and Ecaj_0647 (SEQ ID NO:7), Ech_0905 (SEQ ID NO:8), Ecaj_0647 (SEQ ID NO:7), or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, 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 the subject has an Ehrlichia chaffeensis infection; and (c) administering a therapeutic compound to treat Ehrlichia infection in the subject. 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 multiplex immunoassay, a dipstick test, or a particulate-based assay. The subject may be a dog or a human. The therapeutic compound may be an antibiotic (e.g., doxycycline). The therapeutic compound may be an antibody (e.g., a polyclonal antibody, a monoclonal antibody, a mammalian antibody, or a humanized antibody). The antibody may be present in a multimer.

[0019] Another aspect of the present disclosure relates to an in vitro method of detecting an ehrlichiosis infection in a mammalian subject, comprising: (a) obtaining a biological sample from the mammalian subject, wherein the biological sample is preferably serum or blood; and (b) performing a polymerase chain reaction (PCR) amplification that can selectively expand a nucleic acid encoding Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NO:2), Ech_O35O (SEQ ID NO:3), Ech_0506 (SEQ ID NO:4), Ech_0679 (SEQ ID NO:5), Ech_0494 (SEQ ID NO:6), Ech_0905 (SEQ ID NO:8), Ecaj_0647 (SEQ ID NO:7), or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto; wherein expansion of the nucleic acid indicates that the mammalian subject has ehrlichiosis.

[0020] Yet another aspect of the present disclosure relates to the pharmaceutical composition described above or herein for use in preventing, in prophylaxis of, and / or treating ehrlichiosis or an infection by E. chaffeensis or E. canis in a subject.

[0021] Another aspect of the present disclosure relates to a nucleic acid as defined above or herein for use in preventing and / or treating ehrlichiosis or an Ehrlichia chaffeensis infection in a subject.

[0022] Yet another aspect of the present disclosure relates to a polypeptide for use in preventing and / or treating ehrlichiosis or an Ehrlichia chaffeensis infection in a subject, wherein said polypeptide is Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NO:2), Ech_O35O (SEQ ID NO:3), Ech_0506 (SEQ ID NO:4), Ech_0679 (SEQ ID NO:5), Ech_0494 (SEQ ID NO:6), Ech_0905 (SEQ ID NO:8), Ecaj_0647 (SEQ ID NO:7), or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the sequence set forth in any one of SEQ ID NOs:l, 2, 3, 4, 5, 6, 7, 8.

[0023] Another aspect of the present disclosure relates to the pharmaceutical composition, the nucleic acid, or the polypeptide for the use described above, wherein said pharmaceutical composition, nucleic acid or polypeptide induces an immune response in the subject upon administration to the subject. The immune response may be protective for the subject from ehrlichiosis or an infection by E. chaffeensis or E. canis. Some aspects of the present disclosure relate to the pharmaceutical composition for use described above, the nucleic acid for use described above, or the polypeptide for use described above, wherein said pharmaceutical composition, nucleic acid or polypeptide is to be administered subcutaneously, intramuscularly, nasally, via inhalation or aerosol delivery, or intradermally to the subject. Another aspect of the present disclosure relates to the pharmaceutical composition for use described above, the nucleic acid for use described above, or the polypeptide for use described above, wherein said pharmaceutical composition, nucleic acid or polypeptide is to be administered to the subject in combination with an Ehrlichia bacterin or an adjuvant. The pharmaceutical composition may result in an immune response, preferably a protective immune response, for the subject against ehrlichiosis or infection by E. chaffeensis or E. canis.

[0024] As used herein “immunoreactive protein” refers to a polypeptide that can react with a biological sample from a subject. For example, the immunoreactive protein may react with a serum sample from a mammalian subject, such as a dog or human, that has been exposed to E. chaffeensis or E. canis. The immunoreactive protein preferably can induce an immune responsewhen administered to a mammalian subject (e.g., intravenously, intranasally, or intramuscularly). The immunoreactive protein is preferably an immunodominant protein. The immunoreactive protein is preferably be Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NO:2), Ech_0350 (SEQ ID NO:3), Ech_0506 (SEQ ID NO:4), Ech_0679 (SEQ ID NO:5), Ech_0494 (SEQ ID NO:6), and Ecaj_0647 (SEQ ID NO:7), Ech_0905 (SEQ ID NO:8), and / or Ecaj_0647 (SEQ ID NO:7), or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto. If desired, the immunoreactive protein may comprise additional sequences that may be separately immunogenic or immunoreactive.

[0025] As used herein, the term “polypeptide” encompasses amino acid chains comprising at least 50 amino acid residues, and more preferably at least 75 amino acid residues or at least 100 amino acid residues, wherein the amino acid residues are linked by covalent peptide bonds. As used herein, an “antigenic polypeptide” or an “immunoreactive polypeptide” is a polypeptide 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 polypeptide. An immunoreactive polypeptide may be used in a diagnostic test using a biological sample (e.g., a serum sample) from a mammalian subject, preferably a human or a dog, to determine if the subject has been exposed to E. chaffeensis or E. canis. An antigenic polypeptide may comprise or consist of an immunoreactive sequence derived from an immunoreactive Ehrlichia protein as described herein (e.g. , provided in Table 1 ), and the polypeptide may comprise one or more 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. The antigenic polypeptide or immunoreactive polypeptide is covalently bound to a solid substrate, e.g., in an immunoassay such as a lateral flow test, etc.

[0026] Ehrlichia immunoreactive polypeptides as described herein may be a recombinant polypeptide, synthetic polypeptide, purified polypeptide, immobilized polypeptide, delectably labeled polypeptide, encapsulated polypeptide, or a vector-expressed polypeptide. The Ehrlichia immunoreactive polypeptides provided herein may be truncated or may comprise a deletion mutation, without eliminating the immunoreactivity of the resulting peptide or polypeptide. An immunoreactive peptide or polypeptide disclosed herein may also be comprised in apharmaceutical composition such as, e.g., a vaccine composition that is formulated for administration to a human or canine subject.

[0027] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.

[0028] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” may mean at least a second or more.

[0029] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the inherent variation in the method being employed to determine the value, the variation that exists among the study subjects, or a value that is within 10% of a stated value.

[0030] 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”) arc inclusive or open- ended and do not exclude additional, unrecited elements or method steps.

[0031] The terms “subject,” “host,” “patient,” and “individual” are used interchangeably herein to refer to any mammalian subject for whom therapy is desired, particularly humans and dogs.

[0032] The term “unit dose” when used in reference to a therapeutic composition refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent, z.e., carrier, or vehicle.

[0033] The term “immunotherapy” refers to treatment of disease (e.g., ehrlichiosis) by modulating an immune response to a disease antigen.

[0034] The term “effective amount” is an amount sufficient to effect beneficial or desired clinical results. An effective amount can be administered in one or more administrations. For purposes of this application, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease state. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease.

[0035] An effective response of a patient or a patient’s “responsiveness” to treatment refers to the clinical or therapeutic benefit imparted to a patient at risk for, or suffering from, a disease or disorder. Such benefit may include cellular or biological responses, a complete response, a partial response, a stable disease (without progression or relapse), or a response with a later relapse. For example, an effective response can be reduced tumor size or progression-free survival in a patient diagnosed with cancer.

[0036] “ Treatment” and “treating” refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition.

[0037] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, arc 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 ail from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0039] 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.

[0040] FIGS. 1A-C. Expression of E. chaffeensis and / .. canis proteins by IVTT and immunoreactivity screening by ELISA. (A) Detection of IVTT expression of selected proteins of E. chaffeensis and E. canis by dot immunoblot with anti-His-tag antibody. CTL, the negative control (IVTT reaction without plasmid template). Pooled sera of HME patients or CME dogs were used to screen E. chaffeensis (B) and E. canis (C) proteins, respectively. ELISA OD values represent the mean optical density reading from 3 wells (± standard deviation) after background subtraction. A sample OD of >0.2 was considered positive and >0.5 a strong positive after subtracting negative control (an IVTT reaction with empty plasmid template and a normal human or canine serum control) readings. TRP120 (B) and TRP19 (C) were used as positive controls.

[0041] FIGS. 2A-B. Immunoreactivity of new E. chaffeensis and E. canis immunodominant proteins. (A) Immunoreactivity comparison of 7 E. chaffeensis immunodominant proteins and TRP120 by ELISA. IVTT-expressed proteins were probed with a panel of convalescent sera from 8 HME patients. (B) Immunoreactivity comparison of E. canis immunodominant protein Ecaj_0647 and TRP19 by ELISA. IVTT-expressed proteins were probed with a panel of convalescent sera from 8 CME dogs. OD650 values represent the mean optical density reading from 3 wells (± standard deviation) after background subtraction. A sample OD650 of > 0.2 was considered positive and > 0.5 a strong positive after subtracting negative control (an IVTT reaction with empty plasmid template and a normal human or canine serum control) readings.

[0042] FIGS. 3A-C Conformation-dependent immunoreactivity of E. chaffeensis immunodominant proteins. (A) Immunorcactivity of the denatured IVTT-expressed E. chaffeensis proteins compared with TRP120 by ELISA using a panel of 8 HME sera. (B) Immunoreactivity of overlapping synthetic peptides spanning 2 E. chaffeensis immunoreactive proteins by ELISA with pooled HME sera. Positive control, a TRP120 epitope peptide. OD650 values represent the mean optical density reading from 3 wells (± standard deviation). A sample OD650 of > 0.2 was considered positive and > 0.5 a strong positive after subtracting negative control (A: an IVTT reaction with empty plasmid template and a normal human or canine serum control; B: a negative peptide) readings. (C) Conformation-dependent immunoreactivity of E. chaffeensis proteins by dot immunoblot. Immunoreactivity of the native and denatured proteins and TRP120 was detected with serum from an HME patient. All proteins were IVTT expressed and purified.

[0043] FIGS. 4A-B. Immunoreactivity of new E. canis immunodominant protein. (A) Immunoreactivity of the denatured IVTT-expressed Ecaj_0647 protein compared with TRP19 by ELISA using a panel of 8 CME sera. OD650 values represent the mean optical density reading from 3 wells (± standard deviation). A sample OD650 of > 0.2 was considered positive and > 0.5 a strong positive after subtracting negative control (an IVTT reaction with empty plasmid template and a normal human or canine serum control) readings. (B) Immunoreactivity of native and denatured Ecaj_0647 protein compared with TRP19 by dot immunoblot with pooled CME sera. Both proteins were IVTT-expressed and purified.

[0044] FIG. 5. Quantity analysis of antigenic proteins in E. chaffeensis and E. canis immunomes.

[0045] FIG.6. A schematic diagram of E. chaffeensis / E. canis immunome project showing our identification strategy and the numbers of identified proteins in this study (green) and our previous publications (blue and red) (Luo T, et al. 2020; Luo T, et al. 2021.).DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0046] In some aspects, an immunoreactive polypeptide described herein, preferably Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NO:2), Ech_0350 (SEQ ID NO:3), Ech_0506 (SEQ ID NO:4), Ech_0679 (SEQ ID NO:5), Ech_0494 (SEQ ID NO:6), and Ecaj_0647 (SEQ ID NO:7), Ech 0905 (SEQ ID NO:8), and / or Ecaj_0647 (SEQ ID NO:7), may be used in diagnostic or prophylactic tools for detection of or immunization against Ehrlichia infection. For example, the immunoreactive polypeptides may be used in solution-phase assays, or in assays in which the isolated immunoreactive polypeptide is immobilized on a surface of a support substrate. The immunoreactive polypeptide or an RNA encoding the immunoreactive polypeptide may be comprised in a vaccine formulation to induce an immune response, including a protective immune response, in a subject against Ehrlichia chaffeensis or Ehrlichia canis. One or more immunoreactive polypeptides can 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 or polypeptides may be coupled, conjugated, or cross-linked. The mRNA encoding the immunoreactive polypeptide can be included in a pharmaceutical composition; for example, the mRNA may be chemically modified and comprised in a viral vector (e.g., an adenovirus). In some aspects, the immunoreactive protein is preferably immunodominant or can generate an ELISA OD650 indicating immunoreactivity in a serum sample from a subject, preferably a human or a dog, that has been exposed to E. chaffeensis or E. canis.

[0047] Intracellular bacteria E. chaffeensis and E. canis have relatively small genomes (1.2 Mbp and 1.3Mbp, respectively) that encode less than 1000 proteins (Dunning Hotopp et al., 2006; Mavromatis et al., 2006). Commercial gene synthesis and cloning can facilitate experimental screening of antigenic proteins feasible. High throughput immunomics-based antigen has been used to test undiscovered antigenic proteins from E. chaffeensis and E. canis (Luo et al., 2020; 2021). ANTIGENpro is a sequence-based predictor of protein antigenicity (Magnan et al., 2010) and can be used to guess about the antigenicity of hypothetical polypeptides; however, differences exist between in silico predictions and in vitro or in vivo testing, and a significant amount of unpredictability presently exists for such in silico predictions. About half of the proteins in the E. chaffeensis and E. canis proteome have been screened, and multiple immunoreactive ehrlichialproteins have been identified (Luo et al., 2020; 2021 ). Most of the recently discovered Ehrlichia immunoreactive proteins were predicted to be secreted effector proteins with antibody epitopes that exhibit complete or partial conformation dependence (Luo et al. , 2020; 2021). Comprehensive screening and analysis data provided herein allows for a much more detailed analysis of the antibody-reactive immunomes of E. chaffeensis and E. canis than earlier analyses including identification of E. chaffeensis GroEL as the first antibody-reactive protein in 1993 (Sumner et al., 1993). Immunodominant proteins from the Ehrlichia spp. immunomes reported herein can be used in diagnostic, vaccine, and immunotherapeutic approaches for treatment of human and canine ehrlichiosis.I. Immobilized Immunoreactive Polypeptides

[0048] The immunoreactive polypeptide, preferably from Table 1, may be immobilized onto a surface of a support or a solid substrate; for example, the immunoreactive polypeptide may be immobilized directly or indirectly by coupling, cross-linking, adsorption, encapsulation, or by any appropriate method known in the ail. By way of non-limiting example, binding of an immunoreactive polypeptide 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 250-700 ng or about 450-550 ng. The immunoreactive polypeptide is preferably Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NOG), Ech_0350 (SEQ ID NOG), Ech_0506 (SEQ ID NO:4), Ech_0679 (SEQ ID NOG), Ech_0494 (SEQ ID NO:6), and Ecaj_0647 (SEQ ID NOG), Ech_0905 (SEQ ID NOG), and / or Ecaj_0647 (SEQ ID NOG), or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity.

[0049] The immunoreactive polypeptide can be 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 immunoreactive polypeptide 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 polypeptide (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.

[0050] Numerous support substrates for polypeptide immobilization are known in the art which may be employed with an immunoreactive polypeptide 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.II. Detectably-Labeled Immunoreactive Polypeptides

[0051] An immunoreactive polypeptide (preferably from Table 1) 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 polypeptide may be used, e.g., in diagnostic or prophylactic methods and compositions. The polypeptide portion of the detectably labeled immunoreactive polypeptide may be immobilized on a surface of a support substrate. The detectable label may be used to immobilize the detectably labeled immunoreactive peptide to the surface of a support substrate. The immunoreactive polypeptide is preferably Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NO:2), Ech_0350 (SEQ ID NO:3), Ech_0506 (SEQ ID NO:4), Ech_0679 (SEQ ID NO:5), Ech_0494 (SEQ ID NO:6), and Ecaj_0647 (SEQ ID NO:7), Ech_0905 (SEQ ID NO:8), and / or Ecaj_0647 (SEQ ID NO:7), or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity.

[0052] 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.

[0053] The detectable label may be a photoluminescent probe, such as a fluorophore or a nanoparticle, such as for example a strontium aluminate nanoparticle (e.g., see Paterson et al., 2014). Exemplary labels include, but are not limited to, a particulate label such as colloidal gold, a radioactive isotope such as astatine211,14carbon,51chromium,36chlorine,57cobalt,58cobalt,copper67,152EU, gallium67,3hydrogen, iodine123, iodine125, iodine131, indium111,59iron,32phosphorus, rhcniuml86, rhcniuml88,75selenium,35sulphur, tcchnicium-99, tcchnctium-99m or yttrium90, a colorimetric label such as dinitrobenzene, dansyl chloride, dabsyl chloride, any of the azo, cyanin or triazine dyes, or chromophores disclosed in U.S. Patents 5.470,932, 5,543,504, or 6,372,445, all of which are incorporated herein by reference; a paramagnetic label such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) or erbium (III), a fluorescent label such as Alexa 350, Alexa 430, AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY- TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and / or Texas Red, or Lucifer Yellow, an enzyme label such as urease, luciferase, alkaline phosphatase, (horseradish) hydrogen peroxidase, or glucose oxidase, or a chemiluminescent label such as luminol, phthalazinedione, and others disclosed in any of U.S. Patents 4,373,932, 4,220,450, 5,470,723, and U.S. Patent No. Application 2007 / 0264664, all of which are incorporated herein by reference.III. Methods of Producing an Immunoreactive Polypeptide

[0054] An immunoreactive polypeptide (e.g., preferably from Table 1, or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto) may be produced using in vitro transcription and translation (IVTT) methods, may be recombinantly produced using a variety of cell types (e.g., bacterial cells, mammalian cells, E. coli, yeast, insect cells, etc.), or in some instances may be synthesized (e.g., using solid-phase synthesis). IVTT and synthetic methods can provide certain advantages over recombinant approaches, since the resulting polypeptides can produce highly pure forms without contaminating bacterial or other proteins that might result in false positive reactions when utilizing recombinant proteins. Thus, IVTT and synthetic methods have an advantage of lacking many of the costly and laborious purification procedures often associated with recombinant methodologies.

[0055] A variety of IVTT approaches are known in the art and may be used with the present disclosure. IVTT generally involves cell-free methods for production or synthesis of a protein from DNA. The cell-free system for protein production may use, e.g., E. coli extract, protozoanextracts, yeast extracts, human cell extract, wheat germ extract, mammalian extracts, extracts from cultured human cell lines, rabbit reticulocyte lysate, insect cell extract, or reconstituted and purified E. coli components. A variety of kits are commercially available including, e.g., RTS (FivePrime, San Francisco, CA), Expressway™ (Life Technologies); S30 T7 high yield (Promega), One-step human IVT (Thermo Scientific), WEPRO® (CellFree Sciences), TNT® coupled (Promega), RTS CECF (5 PRIME), TNT® Coupled (Promega), Retie lysate IVT™ (Life Technologies); TNT® T7 (Promega), EasyXpress Insect kit (Qiagen / RiN A), PURExpress® (New England Biolabs), and PURESYSTEM® (BioComber). Such methods can be used to incorporate unnatural amino acids into proteins, if desired. Cell-free expression systems that may be used include those described, e.g., in Zemella et al., 2015.

[0056] An isolated immunoreactive protein as disclosed herein may be produced using an appropriate method known in the organic chemistry arts. For example, peptides may be produced using one of the established solid-phase peptide synthesis techniques. Polypeptides described herein may be synthesized using equipment for automated peptide synthesis that is widely available from commercial suppliers such as Perkin Elmer (Foster City, CA), or the polypeptide may be chemically synthesized using solution-phase techniques such as those described in Carpino et al., 2003 or U.S. Patent No. App. 2009 / 0005535. The polypeptides may be synthesized, e.g., using solid-phase peptide synthesis (SPPS), t-Boc solid-phase peptide synthesis, or Fmoc solidphase peptide synthesis.

[0057] The immunoreactive polypeptide can be recombinantly prepared from a nucleic acid encoding the peptide. Such a nucleic acid may be operably linked to an expression vector. By way of nonlimiting example, an immunoreactive protein may be expressed from a vector and isolated from the growth media of a host cell comprising the vector. The immunoreactive polypeptide may be produced in a cell-free system from a nucleic acid encoding the polypeptide.

[0058] An immobilized immunoreactive protein as disclosed herein may be conjugated, crosslinked, or adsorbed, either directly or indirectly onto a surface of a support substrate. An immobilized immunoreactive polypeptide may be synthesized onto a support substrate.

[0059] It is anticipated that virtually any method of protein or peptide immobilization known in the art which would not impact the structure or function of the disclosed polypeptidesmay be used to immobilize an immunoreactive protein or polypeptide as disclosed herein. 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 sulfo succinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate (sSMCC), N-[maleimidocaproyloxy]sulfosuccinimide ester (sEMCS), N-maleimidobenzoyl-N- hydroxysuccinimide ester (MBS), glutaraldehyde, l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI), Bis-diazobenzidine (BDB), or N-acetyl homocysteine thiolactone (NAHT), and others disclosed in any of U.S. Patents 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. The immunoreactive protein 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 A 12- A 13, 1991).

[0060] The immunoreactive polypeptide 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 tctrachloro-3a-6 oc-diphcnylglycouril-3 attached to the antibody (U.S. Patent No. Nos. 4,472,509 and 4,938,948, each incorporated herein by reference). Proteins, polypeptides and peptides can also be immobilized by coupling to other peptides or to condensation groups immobilized on a surface or present in an immobilization buffer such as glutaraldehyde or periodate. Conjugates with fluorescence markers may also be 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.

[0061] In general, regardless of the method of preparation or immobilization status, the immunoreactive protein is preferably prepared in a substantially pure form. Preferably, the immunoreactive proteins are at least about 80% pure, more preferably at least about 90% pure, even more preferably at least about 95% pure, and most preferably at least about 99% pure.IV. Ehrlichia Vaccine Compositions

[0062] Previous work has shown that Ehrlichial proteins that induce antibody responses can provide protective immune responses; thus, in some aspects an immunoreactive protein provided herein (e.g., preferably from Table 1, or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto) may be included in a pharmaceutical composition such as a vaccine composition for administration to a mammalian or human subject. For example, protection against E. chaffeensis infection has been demonstrated with epitope- specific antibodies directed at OMP and TRPs in in vitro models and in animal models (Kuriakose et al., 2012; Li et al., 2002; Li et al., 2001), demonstrating that ehrlichial proteins that elicit strong antibody responses to linear epitopes are protective. Vaccine compositions provided herein are preferably immunogenic and induce an immune response in a mammalian subject (e.g., a human or a dog). Although the vaccine composition may preferably induce a protective immune response against E. chaffeensis or E. canis, in some instances the immune response induced by the vaccine composition is not protective against E. chaffeensis or E. canis.

[0063] 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 (e.g., Remington, 2012). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the vaccine compositions of the present invention is contemplated. Pharmaceutical compositions that contain an immunoreactive polypeptide provided herein may be an immunoreactive composition that can react with the immune system of a subject or an immunogenic composition that can cause or result in an immune response in the subject. Pharmaceutical compositions that can be administered to a subject to cause an immune response in the subject, preferably to an immunoreactive polypeptide provided herein, are referred to as vaccine compositions. Vaccine compositions preferably resultin an immune response against an immunoreactive polypeptide provided herein, and, although protective immune responses in the subject resulting from the vaccine composition arc not required, it is anticipated that the immune response in the subject may preferably be protective against future infection by E. chaffeensis or E. canis.

[0064] 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 cams.

[0065] 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.B. RNA Vaccines

[0066] In some aspects, a ribonucleic acid (RNA) comprising an open reading frame encoding a polypeptide of Table 1, or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto, is included in an RNA vaccine or pharmaceutical composition. The RNA is preferably a mRNA, and the RNA may be comprised in a lipid nanoparticle (e.g., comprising an ionizable cationic lipid, a neutral lipid, a sterol, and / or a PEG-modified lipid) or a viral vector (e.g., an adenovirus, an adeno-associated virus, etc.).

[0067] RNA vaccines offer a variety of advantages. Since mRNA is a non-infectious, nonintegrating platform, there is no significant risk of infection or insertional mutagenesis. mRNA is degraded by normal cellular processes, and its in vivo half-life can be extended by chemical modifications and delivery methods (Kariko, et al., 2008; Kauffman, et al., 2016; Guan & Rosenecker, 2017; Thess et al., 2015; Kariko et al., 201 1). Immunogenicity of mRNA can also be reduced, if desired, to further increase the safety profile (Kariko, etal., 2008; Thess etal., 2015;Kariko et al., 2011 ). Various modifications can make mRNA more stable and highly translatable (Kariko, et al., 2008; Thcss et al., 2015; Kariko et al., 2011). Efficient in vivo delivery can be achieved by formulating mRNA into earner molecules, which can allow for rapid uptake and expression in the cytoplasm (reviewed in Kauffman, et al., 2016; Guan & Rosenecker, 2017).

[0068] The RNA may be comprised in a variety of formulations, such as nanoparticles and lipid nanoparticles. The RNA or mRNA may be comprised in protamine, a protamine liposome, polysaccharide particle, cationic nanoemulsion, cationic polymer, cationic polymer liposome, cationic lipid nanoparticle, nanoparticle comprising cationic lipid and cholesterol, nanoparticle comprising (cationic lipid, cholesterol, and PEG), or a dendrimer nanoparticle. These different formulations are further discussed, e.g., in Pardi et al., 2018.

[0069] The RNA or mRNA may be chemically modified or unmodified (also called naked RNA). A variety of modifications to the RNA or mRNA can be made, e.g., to extend the half-life of the RNA after injection to a mammalian subject, such as a dog or a human. Modifications to the mRNA that can be made include a 5' cap, 5'- and 3'-UTRs, optimization of the coding region, and / or including the poly(A) tail; these modifications can be used, e.g., to improve intracellular stability and / or translational efficiency. When a 5’cap is not included on the mRNA, the nRNA may include an internal ribosome entry site (IRES) to promote function. Codon optimization can be included to improve translation or reduce endonucleolytic attack. A poly(A) tail may be included in the mRNA to promote stability. Modified nucleotides can be included to inhibit deadenylation. A 3’ UTR can be included to promote proper translation and intracellular trafficking. A 5’ UTR can be included, optionally with or without an IRES, to promote proper translation and intracellular trafficking and to reduce 5’-exonucleolytic degradation. 5’ caps include 5 '-5 '-triphosphate bridge (ppp) (m7GpppN structure) and anti-reverse cap analogues (ARC As; m27,3'-OGpppG). 3 ’-UTR sequences include 3'-UTR of the eukaryotic elongation factor la (EEF1A1) mRNA. Codon optimization can be used to improve translation efficiency; for example, replacing rare codons with synonymous frequent codons improves translational yield. Nonetheless, in some instances, codon optimization is not used; for example, some proteins require slow translation, which is ensured by rare codons, for their proper folding. A variety of modifications are known and can be used as desired (e.g., Sahin et al., 2014)

[0070] Lipid nanoparticles (LNPs) can be used to deliver the RNA or mRNA. LNPs generally include four components: an ionizable cationic lipid, which may promote self-assembly into virus-sized (-100 nm) particles and help endosomal release of mRNA to the cytoplasm; lipid- linked polyethylene glycol (PEG), which may increase the half-life of formulations; cholesterol, a stabilizing agent; and naturally occurring phospholipids, which can support lipid bilayer structure. LNPs can be used for effective in vivo delivery of self-amplifying RNA and non-replicating mRNA. LNPs can be delivered via intradermal, intramuscular and subcutaneous administration have been shown to produce prolonged protein expression at the site of the injection. The magnitude and duration of in vivo protein production from mRNA-LNP vaccines can be affected by varying the route of administration. For example, intramuscular and intradermal delivery of mRNA-LNPs may result in more persistent protein expression than other systemic delivery routes.

[0071] The lipid nanoparticle may comprises 20-60% ionizable cationic lipid, 5-25% neutral lipid (e.g., disteroylphosphatidyl choline (DSPC)), 25-55% sterol (e.g., cholesterol), and 0.5-15% PEG-modified lipid (e.g., PEG-DMG or PEG-cDMA). Lipid nanoparticle formulations are also discussed in US2020 / 0197510 and U.S. 10,702,600 that can be used. LNPs can optionally contain chitosan, cationic 1,2 dioleoyloxy 3 trimethylammoniumpro->pane (DOTAP), dioleoylphosphatidylethanolamine (DOPE), or ionizable dendrimer, if desired.

[0072] The RNA or mRNA may be included in a self-amplifying or replicon RNA vaccine or a non-replicating mRNA vaccine. The RNA or mRNA may preferably be included in a nonreplicating mRNA vaccine. Self-amplifying mRNA (SAM) vaccines typically include portions of an alphavirus genome, wherein genes encoding the RNA replication machinery are included but the genes encoding the structural proteins are replaced with the antigen of interest (e.g., Perri et al., 2003). Preferably, the mRNA is included in a directly injectable, non-replicating mRNA vaccine.

[0073] The RNA or mRNA may preferably be produced via Good Manufacturing Practices (GMP) techniques. GMP production of mRNA typically begins with DNA template production followed by enzymatic IVT. Depending on the specific mRNA construct and chemistry, the protocol may be modified to accommodate modified nucleosides, capping strategies and / or template removal. To initiate the production process, template plasmid DNA (e.g., produced inEscherichia coli.) can be linearized using a restriction enzyme to allow synthesis of runoff transcripts with a poly(A) tract at the 3 ' end. Next, the mRNA can be synthesized from NTPs by a DNA-dependent RNA polymerase from bacteriophage (e.g., such as T7, SP6, or T3). The template DNA can then be degraded by incubation with DNase. The mRNA can then be enzymatically or chemically capped to enable efficient translation in vivo. mRNA synthesis can be very productive, e.g., yielding in excess of 2 gl’1of full-length mRNA in multi-gram scale reactions under optimized conditions. After synthesis of the mRNA, additional purification steps (e.g., microbeads in batch or column formats) can be performed to remove reaction components including enzymes, free nucleotides, and any residual DNA and / or truncated RNA fragments.C. DNA Vaccines

[0074] A pharmaceutical composition comprising a DNA encoding a polypeptide of Table 1 , or a polypeptide having at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto, is included in an DNA vaccine or pharmaceutical composition. DNA vaccines can be delivered via a viral vector, such as an adenovirus or adeno-associated virus (AAV). RNA vaccines in some instances can be preferable over DNA vaccines because RNA vaccines do not require a viral vector and may be less expensive to manufacture.

[0075] A variety of viral vectors can be used. For example, adenoviruses that can be used include those described in U.S. 9,714,435 and U.S. 9,701,718. Adenoviral vectors include AD26 and ChAdOxl (derived from a chimpanzee adenovirus).D. Peptide and Polypeptide Vaccines

[0076] An immunoreactive polypeptide of Table 1, or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto, can be comprised in a vaccine composition and administered to a subject (e.g., a human or dog) to induce an immune response in the subject against an Ehrlichia organism such as Ehrlichia chaffeensis or Ehrlichia cams. It is anticipated that the immune response may be a protective immune response that that may substantially prevent or ameliorate infection in the subject by an the Ehrlichia organism. A vaccine composition for pharmaceutical use in a subject can preferably comprise an immunoreactive polypeptide of Table 1 and a pharmaceutically acceptable carrier. The vaccine composition comprising the immunoreactive polypeptide may be used to induce an immune response, such asa protective immune response, against Ehrlichia chaffeensis or Ehrlichia canis (e.g., in a human or dog subject).

[0077] The vaccine compositions may comprise, for example, at least about 0.1% of an immunoreactive polypeptide (e.g., preferably from Table 1). The immunoreactive polypeptide 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 over a 1-36 week period. Preferably, 3 doses are administered (e.g., at intervals of 3-4 months), and booster vaccinations may be given periodically thereafter.

[0078] A “suitable dose” generally refers to an amount of an immunoreactive polypeptide that, when administered as described above, can result in an immune response in an immunized patient sufficient to reduce the symptoms of or provide some protection against a subsequent exposure to an Ehrlichia organism. The amount of immunoreactive polypeptide present in a suitable dose (or produced in situ by the nucleic acid in a dose) may range 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.

[0079] A vaccine composition of the present invention may utilize a variety of 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 intramuscularly, intradermally, subcutaneously, intravenously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarec tally, topically, intratumorally, intramuscularly, intraperitoneally, subconjunctivally, intravesicularly, mucosally, intrapericardially, locally, orally, intranasally, or by inhalation, injection, infusion, continuous infusion, lavage, or localized perfusion. A vaccine compositionmay 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, 21stEd. Lippincott Williams and Wilkins, 2005, incorporated herein by reference).

[0080] While any suitable carrier known to those of ordinary skill in the art may be employed in the vaccine 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. Patents 4,897,268 and 5,075,109.

[0081] The vaccine composition may be administered by micro structured 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 (e.g., U.S. Patents 5,797,898, 5,770,219 and 5,783,208, and U.S. Patent No. Application 2005 / 0065463). Such a vaccine composition formulated for ballistic particulate delivery may comprise an isolated immunoreactive polypeptide of Table 1 immobilized on a surface of a support substrate. The support substrate may include, but is not limited to, a microcapsule, a microparticle, a microsphere, a nanocapsule, a nanoparticle, a nanosphere, or a combination thereof.

[0082] Microstructures or ballistic particles that serve as a support substrate for an immunoreactive polypeptide disclosed herein may contain a biodegradable material or 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.

[0083] A vaccine composition may comprise an immobilized or encapsulated immunoreactive polypeptide (e.g., preferably from Table 1, or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto) and a support substrate. The support substrate may 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. Patent 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. Patents 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).

[0084] 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 (z.e., ultrasound) which has been used and described in U.S. Patent No. 5,656,016 for enhancing the rate and efficacy of drug permeation into and through the circulatory system; intraosseous injection (U.S. Patent No. 5,779,708), or feedback-controlled delivery (U.S. Patent No. 5,697,899), and each of the patents in this paragraph is specifically incorporated herein in its entirety by reference.

[0085] A polypeptide 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.

[0086] 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 bebrought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., mcthylparabcns, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

[0087] 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 vacuumdrying 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.

[0088] 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. Prolonged absorption of an injectable composition may be facilitated by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.E. Adjuvants

[0089] In some aspects, an immunogenic composition comprising one or more immunoreactive polypeptides (e.g., preferably from Table 1, or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto) also contains an adjuvant. The composition may preferably be a pharmaceutical preparation or a vaccine composition. A variety of adjuvants are known that can be included. For example, adjuvants such as MF59, AS01, AS02, AS03, AS04, Virosomes, CAF01, CAF04, CAF05, Montanide ISA™ 720, or Montanide ISA™ 51 (e.g., Bonam et al., 2017) can be included.

[0090] Any of a variety of adjuvants may be employed in the pharmaceutical compositions and vaccines provided herein to nonspccifically 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.

[0091] The vaccine composition may further comprise an Ehrlichia canis bacterin or Ehrlichia chaffeensis bacterin. Methods that may be used to generate the bacterin include, but are not limited to, treatment of the Ehrlichia with heat, formaldehyde, formalin, bi-ethylene amine, radiation, or beta-propiolactone treatment. It is anticipated that an E. chaffeensis or E. canis bacterin may be inactivated by any suitable method available, such as, e.g., those described in W02005087803, EP2433646, Vega et al., 2007; or Stuen et al., 2015.

[0092] The vaccine composition includes an adjuvant comprising a triterpenoid, sterol, immunomodulator, polymer, and / or Th2 stimulator. For example, the adjuvant may comprise DEAE Dextran, an immunostimulatory oligonucleotide, and oil (e.g., a light mineral oil), wherein the immuno stimulatory oligonucleotide is a CpG containing ODN, and wherein the adjuvant formulation is a water-in-oil (W / O) emulsion. The vaccine adjuvant may optionally comprise an Ehrlichia bacterin (such as a heat-inactivated E. Canis or E. chaffeensis) and / or a chimeric polypeptide. The chimeric polypeptide is preferably a fusion protein comprising multiple antigenic epitopes from different Ehrlichial proteins. The chimeric polypeptide may optionally further comprise a polypeptide from Table 1 expressed in the fusion protein. The immunogenic or vaccine composition may include an antigen component and an adjuvant formulation comprising a saponin (e.g., present in an amount of about 1 pg to about 5,000 pg per dose), a sterol (e.g., present in an amount of about 1 pg to about 5,000 pg per dose), a quaternary ammonium compound (e.g., present in an amount of about 1 pg to about 5,000 pg per dose), a polymer (e.g., present in an amount of about 0.0001% v / v to about 75% v / v.), and an ORN / ODN; the saponin may be Quil A or a purified faction thereof, the sterol may be cholesterol, the quaternaryammonium compound may be dimethyl dioctadecyl ammonium bromide (DDA), the polymer may be polyacrylic acid, and the ORN / ODN may be a CpG. The adjuvant may comprise a glycolipid, such N-(2-deoxy-2-L-leucylamino-P-D-glucopyranosyl)-N-octadecyldodecanamide acetate. The adjuvant may comprise an immuno stimulatory oligonucleotide, a polyacrylic acid polymer and at least two of the following: (a) dimethyl dioctadecyl ammonium bromide (DDA); (b) a sterol; and / or (c) N-(2-deoxy-2-L-leucylamino-P-D-glucopyranosyl)-N-octadecyldodecanamide acetate. For example, the vaccine composition may comprise an adjuvant as described, e.g., in U.S. Patent No. 10,238,736, U.S. Patent No. 8,580,280, or US Publication 2019 / 0008953.

[0093] The vaccine composition may include an adjuvant formulation comprising a triterpenoid saponin, a sterol, a quaternary ammonium compound, and a polyacrylic acid polymer, wherein the antigen component comprises or consists of a Ehrlichia bacterin (such as a heat- inactivated E. Canis). For example, the vaccine composition may comprise an adjuvant as described, e.g., in U.S. Patent No. 9,662,385.

[0094] In some aspects, an immunogenic or vaccine composition as disclosed herein comprises an oil-based adjuvant comprising an Ehrlichia bacterin (such as a heat-inactivated E. Canis or E. chaffeensis). The adjuvant formulation may comprise an immunostimulating oligonucleotide, polycationic carrier, sterol, saponin, quaternary amine, TLR-3 agonist, glycolipid, and / or MPL-A (or an analog thereof) in an oil emulsion. For example, the vaccine composition may comprise an adjuvant as described, e.g., in U.S. Patent No. 10,117,921 or US 2019 / 0038737. The vaccine may further comprise one or more emulsifiers. For example, the vaccine composition may comprise an adjuvant as described, e.g., in U.S. Patent No. 9,545,439 or U.S. Patent No. 8,980,288.

[0095] Adjuvants such as MF59 (e.g., Calabro et al., 2013), AS01 (Didierlaurent, et al., 2014), AS02 (Gargon and Van Mechelen, 2011), AS03 (Morel, S. et al., 2011), AS04 (Didierlaurent, et al., 2009), Virosomes (Kiinzi, et al., 2009), CAF01 (Tandrup Schmidt, et al., 2016), CAF04 (Billeskov, et al., 2016), CAF05 (Billeskov, et al., 2016), Montanide ISA™ 720 (Aucouturier, et al., 2002), or Montanide ISA™ 51 (Aucouturier, et al., 2002) can be used.V. Biological Functional Equivalents

[0096] The immunoreactive polypeptide (e.g., preferably from Table 1, or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto) can preferably bind an Ehrlichia chaffeensis or Ehrlichia canis specific antibody. Determining whether or to what degree a particular immunoreactive polypeptide, or an analog thereof, can bind an E canis specific antibody can be assessed using an in vitro assay such as, for example, an enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immuno staining, latex agglutination, indirect hemagglutination assay (IHA), complement fixation, indirect immnunofluorescent assay (FA), nephelometry, flow cytometry assay, chemiluminescence assay, lateral flow immunoassay, u-capture assay, mass spectrometry assay, particle-based assay, inhibition assay and / or an avidity assay.

[0097] An immunoreactive polypeptide provided herein 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 immunoreactive polypeptide derived from an Ehrlichia 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 immunoreactive polypeptide disclosed herein without appreciable loss of interactive capacity, as demonstrated by detectably unchanged antibody binding. It is thus contemplated that an immunoreactive polypeptide disclosed herein (or a nucleic acid encoding such a polypeptide) which is modified in sequence and / or structure, but which is unchanged in biological utility or activity, remains within the scope of the present disclosure. The immunoreactive polypeptide may have, e.g., at least 90%, at least 95%, or at least 99% sequence identity with a polypeptide of Table 1, and the immunoreactive protein may optionally have 1, 2, 3, 4, 5, or more amino acid substitutions, insertions and / or deletions as compared to a polypeptide of Table 1.

[0098] 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 polypeptides are thus defined herein as those peptides in which certain, not most or all, of the amino acids maybe substituted. Of course, a plurality of distinct peptides with different substitutions may easily be made and used in accordance with the invention. Identification of epitopes in the immunoreactive polypeptides of Table 1 can be determined using site directed mutagenesis, and any amino acid substitutions, insertions and / or deletions are preferably included outside of epitope regions of the polypeptides of Table 1 in order to not adversely affect the immunoreactivity of the resulting polypeptide.

[0099] 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 within an epitope) such residues may not generally be exchanged. It is anticipated that a mutation in an epitope of an immunoreactive peptide or polypeptide disclosed herein could result in a loss of species-specificity and in turn, reduce the utility of the resulting peptide for use in methods provided herein. Thus, polypeptides that are antigenic (i.e., bind anti-Ehrlichia antibodies specifically) and comprise conservative amino acid substitutions are understood to be included in the present disclosure. 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.

[0100] Amino acid substitutions, such as those which might be employed in modifying an immunoreactive polypeptide 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.

[0101] Isoforms of the immunoreactive polypeptides disclosed herein can be used in the compositions and methods provided herein. An isoform contains the same number and kinds ofamino acids as an immunoreactive polypeptide as disclosed herein, but the isoform has a different molecular structure. The isoforms contemplated preferably have essentially the same or the same properties as a polypeptide as described herein.

[0102] Nonstandard amino acids may be incorporated into proteins by chemical modification of existing amino acids or by de novo synthesis of a polypeptide 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, and a variety of nonstandard amino acids are well known in the art.

[0103] The present disclosure contemplates a chemical derivative of an immunoreactive polypeptide 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 polypeptides 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 polypeptides that 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.

[0104] It should be noted that all amino-acid residue sequences are represented herein by formula 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 arc retained by the protein. In keeping with standard protein nomenclature, abbreviations for amino acid residues are known in the art.

[0105] In addition to the biological functional equivalents discussed above, it is contemplated that structurally similar compounds may be formulated to mimic the key portions of an immunoreactive peptide disclosed herein. Such compounds, which may be termed peptidomimetics, may be used in the same manner as immunoreactive peptides disclosed herein and, hence, also are functional equivalents. Methods for generating specific structures are disclosed, e.g., in Mizuno et al., 2017, as well as in U.S. Patents 5,446,128; 5,710,245; 5,840,833; 5,859,184; 5,440,013; 5,618,914; and 5,670,155.VI. Methods of Detecting Ehrlichia infection

[0106] 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 and Walker, 2004; Winslow et al., 2003; Winslow et al., 2000; Yager et al., 2005).

[0107] Accordingly, methods of detecting antibodies that specifically bind an Ehrlichia organism in a sample are also provided. Such a method preferably involves contacting a polypeptide of Table 1 (or an immunoreactive polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto), with the test sample, under conditions that allow peptide-antibody complexes to form, and detecting the peptide-antibody complexes. Here, 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.

[0108] Detection of the immunoreactive polypeptide bound to an Ehrlichia specific antibody (i.e., a peptide-antibody complex) can be accomplished using an enzyme-linked immunoassay (e.g., a sandwich ELISA, or a competitive ELISA), a radioimmunoassay, animmunoprecipitation, a fluorescence immunoassay, a chemiluminescent assay, an immunoblot assay, a lateral flow assay, a flow cytometry assay, a mass spectrometry assay, latex agglutination, an indirect hemagglutination assay (IHA), complement fixation, an inhibition assay, an avidity assay, a dipstick test, or a particulate-based assay. Peptide-antibody complexes may be preferably detected using an enzyme-linked immunoassay, a lateral flow assay, or a particle-based assay.

[0109] 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. The sample may preferably be a blood, serum, or plasma sample obtained from a subject, such as a human or dog.

[0110] Ehrlichiosis caused by an Ehrlichia canis infection in humans presents with flulike 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.

[0111] 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, such as fever, lethargy, loss of appetite, shortness of breath, joint pain and stiffness, and may also include more severe 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 progresses to the chronic stage of disease is often fatal.

[0112] 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 can be 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.

[0113] Accordingly, methods of identifying an Ehrlichia infection in a mammalian subject arc also provided. Such a method may involve contacting a sample from the subject with the isolated immunoreactive polypeptide (e.g., preferably from Table 1, or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto) under conditions that allow peptide-antibody complexes to form, and detecting the peptide-antibody complexes. Here, 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 cams organism. The subject may preferably be 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.

[0114] The terms “subject” and “patient” are used interchangeably herein, and may refer to a mammal, especially a human or a dog. A “subject” or “patient” preferably refers to a mammalian Ehrlichia host (z'.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.

[0115] Without wishing to be bound by theory , the ehrlichial immunoreactive polypeptides 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 ail. 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.

[0116] The immunoreactive polypeptides provided herein can be used in methods for determining whether a subject has been immunized against Ehrlichia or is actively infected withan Ehrlichia organism. The method may comprises contacting a sample from the subject with at least one isolated immunoreactive polypeptide (e.g.. preferably from Table 1, or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto) that is not a component of an Ehrlichia vaccine, and detecting whether an antibody in the sample specifically binds to the isolated ehrlichial immunoreactive polypeptide. According to the method, if an antibody in the sample specifically binds to the isolated ehrlichial immunoreactive polypeptide, then this result indicates the subject has or has had an active Ehrlichia infection, and if an antibody does not specifically bind to the isolated ehrlichial immunoreactive peptide, then the subject has either been previously immunized with an Ehrlichia vaccine or is not infected with an Ehrlichia organism. The Ehrlichia organism may be an E. chaffeensis organism or an E. canis organism.

[0117] The immunoreactive polypeptide may be used to bind an Ehrlichia- specific or E. chaffeensis-specific antibody using a variety of methods or kits. The specific binding between an antibody and an Ehrlichial polypeptide as disclosed herein may therefore be assessed by any appropriate method known in the art including, but not limited to, an enzyme-linked immunosorbent assay (ELISA), a sandwich ELISA, a competitive ELISA, immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immuno staining, latex agglutination, indirect hemagglutination assay (IHA), complement fixation, indirect immnunofluorescent assay (FA), nephelometry, flow cytometry assay, chemiluminescence assay, lateral flow immunoassay, u- capture assay, mass spectrometry assay, particle-based assay, inhibition assay and avidity assay. Exemplary methods of detecting the binding of an Ehrlichia-specific antibody to an ehrlichial immunoreactive polypeptide as disclosed herein may include, for example, an ELISA performed in a microplate, a lateral flow test performed using a dipstick or lateral flow device, or a particulatebased suspension array assay, e.g., performed using the Bio-Plex® system (Bio-Rad Laboratories, Hercules, CA, USA).B. ELISA

[0118] The detection of a peptide- antibody complex described herein may preferably be accomplished using an enzyme linked immunosorbent assay (ELISA). This assay may be performed by first contacting an immunoreactive polypeptide (e.g., preferably from Table 1, or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto) 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 samples can then be 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.

[0119] The detection reagent may contain a binding agent (such as, for example, Protein A, Protein G, immunoglobulin, lectin, or free antigen) conjugated or covalently attached to a reporter group or label. Exemplary reporter groups or labels include enzymes (e.g., 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, CA; and Pierce, Rockford, IL).

[0120] The presence or absence of Ehrlichia specific antibodies can 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 is based on or reflects the average mean signal obtained when the immobilized ehrlichial immunoreactive peptide is incubated with samples from an uninfected subject. The cut-off value may be determined using a statistical method or computer program.C. Lateral Flow Tests

[0121] 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 preferred for identifying an E. chaffeensis or E. canis infection at the point of care, which can be particularly importantwhen the subject is, for example, a human or dog exhibiting detectable antibodies during the treatable acute phase of infection.

[0122] 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 veterinarian 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 (e.g., any of U.S. Patents 7,344,893, 7,371,582, 6,136,610, and U.S. Patent No. Applications, 2005 / 0250141 and 2005 / 0047972, or Koczula et al., 2016). 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 or blood sample, and may be drawn laterally from the sample zone to the binding zone which comprises an immunoreactive polypeptide (e.g., preferably from Table 1, or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto) immobilized to a surface of the lateral flow device. In this example, the binding of the immobilized ehrlichial immunoreactive polypeptide on the lateral flow device (e.g., by an antibody or antibodies from a blood or serum sample from the subject) is an indication that Ehrlichia specific antibodies are present in the sample from the subject, indicating an Ehrlichia infection in the subject, such as an E. chaffeensis or E. canis infection in the subject.

[0123] The 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 ehrlichial 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. Peptide-antibody complexes bound to detection reagent may then be detected, as appropriate for the detection reagent used (e.g., based on the presence or absence of a visibly detectable color or fluorescent label, a nanoparticle, a luminescent rare earth nanoparticle, a luminous nanoparticle, a strontium aluminate nanoparticle (e.g., see Paterson et al., 2014; and Wang et al., 2017, etc.). Detection of binding of an antibody from a biological sample from thesubject with the immunogenic protein can be observed, e.g., by the binding of a labeled anti-human antibody or a labeled anti -dog antibody to the antibody bound to the immunoreactive polypeptide.

[0124] A flow-through format ELISA may be performed in which one end of the membrane to which the immunoreactive peptide (e.g., preferably from Table 1) 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 may migrate along the membrane through a region (i.e., a labeling zone) comprising the detection reagent, and flows to the area (i.e., a binding zone) comprising the immobilized ehrlichial immunoreactive peptide. An accumulation of detection reagent at the binding zone indicates the presence of Ehrlichia specific antibodies in the sample.

[0125] 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 typically indicates a negative result. It is within the ability of an ordinarily skilled artisan to select an amount of the immunoreactive polypeptide for immobilization on the membrane that can generate a visually discernible pattern when the biological sample contains a level of antibodies that would be sufficient to generate a positive signal in a standard format ELISA. Preferably, the amount of peptide immobilized on the membrane ranges from about 25 ng to about 1 mg.D. Particulate-Based Assays

[0126] 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 multiplex particle-based assay such as the suspension array Bio-Plex® assay system available from Bio-RadLaboratories, Inc. (Hercules, CA) and Luminex, Inc. (Austin, TX) may be useful in identifying Ehrlichia antibodies in a sample.

[0127] The isolated immunoreactive polypeptide (e.g., preferably from Table 1, or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto) can be immobilized on a surface of a particle for use in a particle-based immunoassay. As described herein, methods of peptide immobilization onto support surfaces are well known in the art. The immunoreactive polypeptide can be labelled as described above and immobilized onto a surface of a particle, and the peptide-particle complex can then preferably be used in an ELISA or flow cytometry assay according to established protocols.VII. Ehrlichia Detection and Vaccination Kits

[0128] One or more immunoreactive polypeptides (e.g., preferably from Table 1 , or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto) can be included in a kit for the detection of antibodies in a sample that specifically bind an Ehrlichia organism, such as E. chaffeensis or E. canis. The kits may thus be used for the diagnosis or identification of an Ehrlichia infection in a subject. The kit may be used for determining whether a subject has been immunized against Ehrlichia or is actively infected with an Ehrlichia organism. Kits are also provided herein for vaccination against or producing an immune response against E. chaffeensis and / or E. canis.

[0129] The kit may be used to perform a method disclosed herein. For example, the kit may be suitable for detecting Ehrlichia antibodies in a sample, for identifying an Ehrlichia infection individual, 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. The immunoreactive polypeptide (e.g., preferably from Table 1, or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto) are preferably comprised in the kit in a pharmaceutical composition. The ehrlichial immunoreactive polypeptide in the kit may be detectably labeled or immobilized on a surface of a support substrate also comprised in the kit. The immunoreactive polypeptide(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 multiwell 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, the 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. Patents 5,797,898, 5,770,219 and 5,783,208, and U.S. Patent No. 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. The labeling or detection reagent may be selected from a group comprising reagents used commonly in the art and including, e.g., 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. A kit may comprise 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] The kit may be used for detecting anti- Ehrlichia antibodies in a sample which may also be used for identification of an Ehrlichia 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 immunoreactive polypeptides (e.g., preferably from Table 1, or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto), and the peptides may be delectably labeled and immobilized to one or more support substrates comprised in the kit.

[0134] The kit may preferably comprise the immunoreactive polypeptide(s) immobilized to one or more separate lateral flow assay devices, such as a nitrocellulose test strips. Each of the test strips may further comprise a detection reagent, for example, a chromophore-labeled protein A. The 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. The solvent may be a sterile, pharmaceutically acceptable buffer and / or other diluent. The 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.IV. Examples

[0137] The following examples arc 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 examples which follow represent techniques discovered by the inventor 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 - Identification of Immunodominant proteins

[0138] E. chaffeensis and E. canis immunomics-based screening. Previously, the predicted E. chaffeensis (Arkansas strain) and E. canis (Jake strain) ORFs in both databases of Integrated Microbial Genomes (IMG) and GenBank were analyzed. After RNA genes, pseudogenes and short ORFs (coded proteins < 42 aa) were excluded, the total number of ORFsin E. chaffeensis and E. canis genome was determined to be 882 and 928, respectively (Luo et al., 2021). The predicted antigenicity of all proteins was determined using ANTIGENpro and respective antigenicity scores obtained (between 0 and 1). The immunoreactivity of all proteins distributed in the top 350 (with antigenicity scores > ~0.6) were previously investigated, excluding previously characterized antigens (such as TRPs and OMPs). In addition, hypothetical proteins (including proteins with domain of unknown function [DUF]) regardless of ANTIGENpro rank were also previously prioritized (Luo et al., 2020; 2021) (FIG. 6).

[0139] To define the complete antigenic repertoire of E. chaffeensis and E. canis, this example further investigated the remaining proteins (n = 444 and n = 405, respectively) that were not examined in previous studies. Proteins were expressed in the cell-free IVTT system, and the expression was confirmed by dot blot of randomly selected proteins (n = 22) from both E. chaffeensis and E. canis (Figure 1A). Since ELISA plate wells can be saturated by IVTT expressed proteins, the differences in expression levels did not influence the relative immunoreactivity between different proteins as established in previous investigations (Luo et al., 2020; 2021).

[0140] The E. chaffeensis and E. canis proteins (n = 444 and n=405, respectively) were screened for immunoreactivity by ELISA using pooled convalescent HME or CME sera, respectively, which had indirect fluorescent- antibody assay (IFA) titers of 1600. A total of 196 (44%) E. chaffeensis and 43 (11 %) E. canis proteins reacted with pooled sera (mean optical density at 650 nm [OD65o] > 0.2 with background subtracted). All E. chaffeensis and E. canis proteins were ranked according to ELISA OD value (from high to low) and are listed in Tables SI and S2, respectively. The E. chaffeensis (n = 40; mean OD650 > 0.8) and E. canis proteins (n = 31; mean OD650 > 0.3) that exhibited the strongest immunoreactivity with pooled sera are shown in Figures IB and 1C.ranked by sseaii ELISA QD vafejes.Eds sag Meao tUSA MKHOIVvEsh tag Mean ELISA Aritigeaidtv e' no. " ODss® score ' no. “ OD^ score79 6095 0 65 008 120 9940 048 0 35SO 0621 0.05 0.38 121 0732 0.48 0.5581 0227 0.64 0.42 122 0700 / 0713 0.47 0.16S2 014S 0 64 0 15 123 0383 047 0 2683 0780 0.64 0.05 124 0174 0.47 0.4284 0598 0.63 0.20 125 0063 0.46 0.15S5 10SO 0.63 0.06 126 1044 0.46 0.068§ 0234 0.63 0.30 127 0977 0.46 0.5387 0588 0.62 0.59 128 0552 0.45 0.0688 0294 0.62 0.22 129 3557 0.45 0.2789 0264 0.62 0.48 130 0073 045 0.5490 0660 0.61 0.45 131 0307 0.45 0.1 1§1 0779 0.60 0.25 132 0844 045 0.1392 0620 0.60 0.60 133 0644 0.45 0.1893 1068 0.59 0.05 134 0643 0.44 0.5594 0212 0.59 0.06 135 0127 044 0.2395 0905 0.57 0.27 136 1077 0.44 0.1096 0521 O.57 0.10 137 0323 0.44 0.1597 0029 0.5? 0.13 138 0172 0.44 0.5498 0337 0.57 0.03 139 0841 0.44 04899 0575 0.56 0.18 140 0338 0.43 0.31100 6401 0.56 0.53 141 OSOl 0.43 0.22101 W? 0.56 0.50 142 0191 0.43 0.65102 0786 0.55 0.03 143 0782 0.43 0.35163 101? 0.55 0.12 144 0522 8.42 0.30104 0269 0.54 0.05 145 0553 0.42 0.14105 0438 0.54 0.07 146 1087 0.41 0.06206 0902 0.53 0.55 147 0581 0.41 0.68107 6890 0.53 0.22 148 0765 0.43 0.41108 0675 0.52 0.48 149 0382 0.39 0.10119 0435 0.52 0.13 150 0483 0.39 0.33HO 0128 0.51 0.45 151 0183 0.39 0.32111 0125 0.51 0.25 152 0892 0.39 0.42112 6328 0.51 0.10 153 0548 8 39 0.28113 6393 0.50 0.08 154 0741 8.39 0.52114 104? 0.50 0.05 155 0?28 0.38 0.49115 6636 0.50 0.12 156 1612 0.38 0.43116 0321 0.50 0.11 15? 0849 0.3? 0.34117 9338 0.50 0.25 158 9492 0.3? 0.18118 0035 0.49 0.21 159 0554 0.37 6.11119 0667 0.49 G.09 160 1079 0 37 0 16Ech_ tag Meas ELISA AithgmidtyEch_ tag Mea® ELISA Aahgeaidty sis. score iso. OtW score1 0452 038 0.21 202 1028 0.19 0052 1084 038 0.27 203 9333 0.18 0233 0885 0.35 0 23 204 0447 0.IS 0.154 0740 034 0 34 203 1115 0.18 0435 8737 0.34 0.23 208 0616 8.1? 0.116 0590 0.34 0.09 20? 0686 8.1? 0.447 0845 034 037 208 10SS 0.17 0 518 050S 0.33 0.39 209 8516 8.18 0199 1082 033 0 14 210 8562 8.16 0.290 8I4§ 032 008 211 8510 0.16 0.551 69 IS 0.32 0.07 212 8599 8.16 0372 0965 032 036 213 0266 0.16 0.163 1070 031 0.20 214 8392 8.15 0 294 0474 0.31 0.06 215 8288 8 15 0865 0093 031 036 216 0530 0.15 0.046 0685 0.29 0.07 217 8169 8.15 0 227 1098 0.22 046 21S 8123 0 14 0.45S 0982 0.28 0.19 219 395( 0.14 0 169 0910 0.28 9.07 220 9334 0.14 0,450 0085 0.28 0 19 221 8311 0.14 0 231 0504 0.28 0.43 222 107S 0.13 0212 0289 0.27 0.45 223 8537 8.13 8 323 0299 0 26 0 26 224 8031 8 13 0 364 0816 0.28 0.05 225 1093 0.13 0045 0313 0.26 0.15 226 8305 8.13 0366 0496 025 0 1? 227 0056 6 13 0237 8036 0.25 8.2? 228 8173 0.13 0.398 0341 0.25 0.25 229 8571 8.13 0.219 8882 0.24 040 230 1106 0.13 0.140 0573 0.23 0.41 231 9369 8. 13 0421 0838 0.22 0.06 232 8702 8.12 0 142 8055 0.22 041 233 0175 0.12 0.443 0724 0.21 0.40 234 8091 0.12 0 144 0048 0.21 043 235 8751 0.12 0.225 0974 0.21 0 23 236 8300 0.12 0.136 0469 0.21 0.16 237 8637 0.12 0 36? 0161 0.19 0.59 238 8S52 8.12 0.338 8909 0.19 0.04 239 8761 0.12 0 399 0536 0.19 0.14 240 1122 0.12 0250 1048 0.19 0.04 241 8439 8.12 0.371 109? 0.19 051 242 8633 0.11 0.28Ech_ tag Meas ELISA Antigenicity Edi_tag Mean ELISA Antigesidty*ci' no. OD«s scorei'°' isc.score243 0217 0.11 834 284 0347 805 025244 8655 O i l 840 285 0746 8.05 040245 8455 0.11 0.45 286 1090 9.04 0.37246 0062 0.11 831 287 0555 804 067247 0466 Oil 0.15 288 0135 0.04 0.4724S 8629 0.11 8.26 2.89 0958 9.04 0.05249 6605 0 11 842 290 0361 804 0 85258 8465 0.11 9.41 291 0362 0.04 0.43251 9211 0.11 8.12 292 0532 804 0.46252 0315 0.11 0.44 293 0788 8.84 0 4?253 89W 0.10 031 294 0858 9.04 038254 0701 0.19 0.4? 295 0567 8.03 0.41255 8132 0 10 9 17 296 0824 003 033256 1005 9.10 9.26 29? 036? 0.03 0.29257 1096 0.19 0.3? 298 0495 8.03 0.3?258 020&W4 0.10 9.1§ 299 0820 0.03 0 16259 099S 0.19 0.41 380 0952 8.03 0 212(50 8543 9.10 9.57 301 0832 0.03 0.36261 0693 0.10 8.29 302 0804 0.02 037262 OS 14 0.19 0.24 383 0931 802 0.39263 0561 0.10 836 304 0771 0.02 0.16264 0774 009 0 13 385 0434 8.01 0 37265 0308 0.09 0.05 386 0400 8.01 0.8?266 06S8 0.09 935 307 80S? 0.01 0.59267 0137 0.09 9.13 388 0899 0 0.18268 0772 0.09 8.07 309 0794 9 0.60269 0339 0.09 8.18 310 0210 & 059278 8969 0.09 9.42 311 0376 0 0.59271 0018 0.09 8.49 312 0538 & 059272 834$ 0.09 9.36 313 0961 0 0.5S273 8789 0.08 9.44 314 0061 & 0.58274 961:8 0.08 8.29 315 0703 & 058275 0032 0.08 9.49 316 0996 8 0.58276 9024 0.08 837 317 0381 & 05727? 9967 0.07 8.59 318 0387 0 057278 8344 0.07 9.17 319 0330 0 0.57279 0237 0.07 8.44 320 0479 & 057288 8124 0.06 9.37 321 0160 0 0.57281 8811 0.06 9.44 322 0783 3 0.57282 0013 0.05 0.42 323 0133 S 0 5?283 1080 0.05 9.28 324 0208 J 0.57„ Edi_tag Mean. ELISA Aetigeasidty Ech_tag Meas ELI SA Antigenicity1 :0' is?. OD&TO score1 ano. CD®-; score325 08® 0 0.% 366 005§ 0 0.47326 0497 0 « :c 367 0020 0 0.47327 05H 0 0.55 368 0001 0 0.47328 0134 0 655 36$ 0651 0 0.47329 0038 0 0.55 370 9455 0 0.4?330 0773 0 0.54 371 0502 0 0.46331 0?91 G 0.54 372 0528 0 0.46332 0913 0 0.54 3?3 0542 0 0.44333 0860 0 0.54 374 0449 0 0.43334 0540 0 0.54 375 0805 0 0.43335 02:98 0 053 376 0082 0 0 43336 1156 G 0.53 37? 0003 0 0.43337 0225 0 053 378 0766 0 0.41338 0045 0 053 379 6473 0 040339 0726 0 0.53 380 0754 0 0.39340 11 16 0 0 52 3S1 1105 0 0.39341 0322 0 0.52 382 0541 0 0.38342 0877 0 652 3S3 090S 0 0.38343 1117 0 0.52 384 0363 0 0.37344 0026 0 0.52 385 0803 0 0.37345 0544 0 0.52 386 0343 0 0.35346 0957 0 0.52 337 8476 0 0.34347 6683 0 051 388 0429 0 0 34348 0119 0 0.51 389 1107 0 0.33349 0066 0 059 390 0595 0 0.33350 0060 0 0.50 391 1064 0 0.32351 $092 0 0.50 392 0448 0 0.31352 0342 0 0 50 393 0935 0 0.31353 S5® 0 0.50 394 9015 0 0.31354 0873 0 0.50 395 0S2S 0 0.31355 1149 0 0.50 396 0928 0 0.30356 1051 0 0.49 39? 9642 0 0.30357 0195 0 0.49 398 0S21 0 0.29358 0793 0 0.49 399 9324 0 0.29359 0464 0 049 400 9162 0 0 29360 0404 0 0.49 401 0512 0 0.29361 0202 0 0:48 402 9005 Q 0.29362 0515 0 0.48 403 0477 0 0.29363 0030 0 0.48 404 9214 9 0.28364 0626 0 0.48 405 1009 0 0.28365 0139 0 0.48 406 9097 0 0.27NEch_ tag Mem ELISA Ai^igeisrity Ech_ tag Meat- ELISA Astigemdly*aiKj. CXEfej score1'J5' no. G©s;sj scare407 0730 0 0.27 426 0520 0 0.1740S 0310 0 0.27 427 0356 0 0 15400 0400 0 f: 77 42S 10® 0 0.1$410 0131 0 0.2§ 420 0823 0 0 14411 0104 0 0.25 430 0403 0 0.13412 0103 0 0.25 431 1082 0 0.13413 0221 0 0.23 432 9831 0 0.13414 0702 0 0.23 433 0714 0 0.12415 1108 0 0.22 434 9290 0 0.12416 0874 0 0.22 435 sms 0 0.11417 00® 0 0.23 436 9930 0 0.11413 0843 0 0.21 437 m? 0 0.10410 0003 0 0.21 433 9077 0 0.10420 1072 0 0.20 439 9917 0 0.10421 0400 0 0 20 440 9584 0 0.®422 0229 0 0 19 441 9511 0 007423 0641 0 0.18 442 1042 0 O.Q8424 1010 0 0.17 443 9389 0 O.OS425 014© 0 0 17 444 1043 0 003TABLE S2 IL protein irErmaseseaciivity ranked by mean ELISA QD values.£^aj_ Mean ELISA Antigenicity Ecaj_ Mean ELISA AntigenicityLag an. ODtee scare *v" tag an. ODsss score1 0274 224 9.42 40 8453 0.22 0.152 064? 1.65 9.57 41 OSO? 0 21 0.353 0026 1 57 0.83 42 0085 0.20 0.234 0642 1.26 0.46 43 03915 0.20 0.085 0710 1 23 0.66 44 0522 0.20 0.506 0943 123 9.46 45 0529 0.19 0.387 0873 0.93 0.06 46 0032 0.19 0.278 0305 092 9.37 47 0039 0.18 0.599 0244 0.88 9.17 48 0536 0.18 0.1416 0817 0.83 0.44 49 0559 0.18 0.1211 9910 0 74 9.33 50 0006 0.17 0.5312 0862 0.67 9.25 51 069? 0.17 0.0913 0293 0 62 9.29 52 0869 0 16 0.0714 0614 0 60 9.60 53 0079 0 16 0.5415 0794 0.47 9.57 54 0677 0.15 0.1216 0534 045 9.10 55 0S74 0 15 0.4217 03910 0.45 0.0? 56 9066 0.15 0.91I S 0820 0.45 0.30 5? 0840 0.15 0.1119 0328 044 9.35 58 0090 0.14 0.6920 0560 0.43 9.06 59 0501 0 14 0.9921 9918 042 9.82 60 OSOO 0.14 0.5722 0809 0.42 9.44 61 0778 0.14 0.45 23 05075 0.36 9.15 62 0452 0 14 0.1324 0848 0.34 9.79 63 0841 0.13 0.0525 6163 034 9.04 64 0544 0 13 0.2626 0515 034 9.33 65 0785 0 13 0.5327 0076 0.33 0.34 66 0864 0.13 0.2228 0879 033 9.40 67 0039 0 12 0.0829 03920 0.33 0.86 68 0215 0.12 0.2430 0038 0 32 9.46 69 0629 0.12 0.3531 0557 030 9.06 70 0531 0.12 0.3432 0034 029 9.23 71 0883 0 12 0.1633 0317 02§ 9.24 72 0184 0.12 0.3034 0962 026 9.87 73 0495 0.11 0.6235 008S 0.26 9.73 74 0205 0 11 0.2936 0533 0.26 9.29 75 0019 0.11 0.6937 0814 0.26 0.30 76 0780 0 11 0.273S 0704 023 9.14 77 0549 0 11 0.5439 0294 0 23 9.06 78 0541 _ 0.10 _ 6.57Eeaj_ Mean ELISA AstigeindtywEcaj:_ Mesa ELISA Aniigenidty tag si<s. ODfe^ scare tag so. QDs$s score9 W44 0.10 0.67 120 0075 0 06 0.430 0202 0.10 0.31 121 0852 0 06 0.161 0045 0.10 0.73 122 0546 6.06 0.-42 0073 0 10 G.S9 123 0896 0 06 0.153 0S86 0 10 0.32 124 0692 0.06 0.284 0586 0.10 0.13 125 0835 0.06 0.2.25 0211 0 10 0.08 126 0759 0 06 0.146 0082 0 10 0.11 127 0908 0.06 0.45? 0 / 81 0.09 0.40 128 0841 0.06 0.998 0561 0.00 0.28 129 0046 0.06 0.4:29 0654 009 0.17 130 0760 0.06 0.380 0813 0.00 0.27 131 0519 0.66 0.071 0521 009 0.54 132 0570 0.06 0.132 0036 009 0.81 133 0642 0 06 0 193 0189 0.09 0.09 134 0894 0.66 0.784 0224 009 0.26 135 0701 0 06 0.555 0875 0.09 0.40 136 0868 0 06 0906 0846 0.09 0.58 137 0627 0.06 0.39? 0652 OOS 0.53 138 0661 0 05 0.318 0631 008 0 30 139 0708 0.95 0.399 0621 0.00 O.?9:140 0670 0 05 0.150 0806 0.0S 0.49 141 0885 0 05 0.21 1 0449 008 006 142 0313 0.95 0.202 0640 0.08 0.47 143 0567 0 05 0.393 0867 008 0.24 144 G532 0.0? 6.1 14 0549 008 042 145 QS01 0.95 0.375 6070 0.08 0.50:148 0871 0.05 0.236 0OS9 008 0.39 147 G55S 0.03 0.137 0556 0.08 0.11 148 0275 0.05 0.288 0816 007 0.31 149 0573 0.05 0.369 W 007 0.27 150 0825 0.05 0.4®0 0093 0.07 0.53 151 0926 0.95 0.S2 1 0552 0.07 0.06 152 0681 0 05 0.292 0518 0.97 0.36 153 0892 0.05 0.503 0585 0.07 0.04 154 0686 0.6? 0.404 0712 0.07 0.58 155 0037 0 05 0.145 0689 007 0.24 156 G665 G.05 0.326 0784 007 0.31 157 OOS7 0 05 0.167 0885 0.07 0.03 158 0256 0 05 0 848 0699 097 0.13 159 G526 G.05 0.469 6713 0.06 0.18 160 0574 0.94 0.21Ecaj_ Kfean EUSA Asiigerscity Ecaj_ Mean ELISA Antigemdtytag ns. ODsw score teg s®. ODw score1 66S8 0.64 0.52 202 0376 6.03 0.492 6842 0.04 9 / 06 203 0776 0.03 0.323 8668 0.64 0.15 264 0092 0.02 0.47 6709 0.04 0.17 205 087? 0.02 0.055 0774 004 0.54 206 0043 0.02 0516 08M 0.04 0.11 207 0243 0.02 0.477 0891 0.64 0.35 203 0362 0.02 0398 0876 004 0.37 209 0621 0 02 0419 0639 (KM 0.33 210 0055 9.02 0.350 0061 (KM 0.56 211 0995 0.02 0.071 0®1 004 045 212 6927 0 02 0.212 8086 (KM 0.27 213 0872 0.02 0.073 9645 0.84 0.22 214 0935 6 02 0.124 0031 064 034 215 9896 0.02 0.865 8074 (KM 0:25 216 W8 0.02 0.266 9635 0.84 0.61 217 0250 0 02 0.187 8928 (KM 0.62 218 0490 0.02 0K48 0§O3 0.64 0.21 219 0893 0.02 0.419 9666 0.84 0.54 220 0053 6 02 0.510 8684 0 64 0.25 221 0024 0.02 0.201 9792 004 0.05 222 0396 0 02 0.362 9818 0.84 0.17 223 0757 6 02 0.043 8663 0 64 0.82 224 8339 002 0.104 6683 064 0.53 225 0815 6 02 0.455 8626 0.84 0.3S 226 8934 602 0.616 6888 0.03 0.23 227 0221 002 0.467 9003 003 0.67 228 0565 6 02 0.148 0575 0.83 0.17 229 0795 802 0.449 6029 0.03 0.35 230 0196 6.02 0.100 0083 0.03 0.21 231 03S0 0.02 0491 9779 0.83 0.06 232 0871 6.01 0.932 6207 0.03 0.53 233 0100 8.01 0.893 8855 0.03 0.24 234 8793 8.01 0064 8099 0.03 0.94 235 0825 9.01 0.115 6624 0.03 0.18 236 0052 6.01 0.256 0659 063 0.44 237 0699 6 01 072? 0940 0.63 0.55 238 0592 6.01 0.208 0672 0.83 0.45 239 0327 6.0! 0.509 ®)91 063 082 240 0465 6 01 0.10G 0706 0.03 0.70 241 0265 8.01 0.061 0085 0.03 0.88 242 0208 6 0! 0.53tagsKS. QDBS score tag so. 0D«.® score3 00® 901 0.24 284 0050 0 0.054 0238 0.01 0.31 285 0051 0 0.225 0057 0.01 0.17 280 0054 0 0.646 0022 0.01 0.S3 28? 0056 0 0.39? 0064 0.01 0.48 288 0058 0 0:098 0001 0.01 0.29 2S9 0059 0 0.429 0040 0.01 0.42 290 00® 0 0.840 0223 0.01 0.1? 291 0063 0 0.941 0203 0.01 0.40 292 0065 0 0.872 0514 0.01 0.14 293 9367 0 0.923 0587 0.01 0.37 294 0069 0 0.944 0929 0.01 0.44 295 ®'S 0 0.205 0193 0.01 0.39 296 0081 0 0:996 0276 091 0 56 29? 0084 0 0: 557 0204 0.01 0.29 29® 0131 0 0.268 0235 0.01 0.39 299 0190 0 0.109 0359 0.01 0.42 300 0197 0 0.130 0765 0.01 0.69 301 02® 3 0.231 0455 0 0.29 302 022? 0 0.552 0080 0 0.26 303 0241 0 0273 (AM 0 0..52 304 0245 0 0 324 0097 0 0.35 305 0251 0 0 035 0218 0 0.38 306 0253 0 0.746 0072 0 0.90 30? 025? 0 0417 0077 0 0.58 308 0262 0 0.318 0473 0 0 26 309 0263 0 0: 119 0344 0 0.50 310 0264 0 0050 0944 0 0.62 311 0267 0 0.281 0002 0 0.04 312 0269 0 0:372 0004 0 0.64 313 0270 0 0.573 0007 0 0.10 314 0272 0 0.334 0011 0 0.24 315 0281 0 0: 535 0012 0 0.73 316 0283 0 0.336 0013 9 0.62 317 0284 0 0: 147 0014 9 0.19 318 0286 0 0.268 0017 0 0.72 319 0292 $ 0.479 9020 9 0.94 329 0295 0 0:950 0023 0 0.42 321 0296 0 0.411 0033 0 0.65 322 0297 0 0.812 6035 0 0.17 323 0301 6 0:913 0048 0 0.55 324 0302 0 0.26Ec3j_ Meas ELISA Aahgeaicity Ecaj_ Mean ELISA Aaligenicity tsg sis. OD® scorei" '' isg sis. ODw store 0303 0 0.23 366 0498 0 0.24 0304 0 0.83 347 0541 0 0.20 0307 0 0.10 36® 0510 0 0.50 0319 0 0.55 369 0511 0 0.0® 0320 0 0.46 370 8550 0 0.30 0322 0 0.29 371 0552 0 0.51 0324 0 0.3® 372 0582 0 0.10 0329 0 0.25 373 0543 0 0.05 0352 0 0.54 374 0616 0 0.28 0353 0 0.1S 375 06 IS 0 0.35 0354 0 0.32 375 0619 0 0.15 0369 0 0.25 377 0650 0 0.77 0364 0 0.37 378 0674 0 0.3® 0367 0 0.4® 379 0790 0 0.21 0370 0 0.57 380 0707 0 0.19 0371 0 0.36 381 9798 0 0.47 0375 0 0.42 332 0811 0 0.07 03 SI 0 0.79 3S3 6854 9 0.38 0389 0 0.17 384 0851 0 0.25 0392 0 0.34 3S5 08® 0 0.26 0393 0 0.31 386 0880 0 0.05 0397 0 6.41 387 0884 0 0.56 0398 0 0.24 388 0921 0 0.55 0402 0 0 52 389 0933 0 037 0403 0 0.43 390 0937 9 0.04 0405 0 0.57 391 6939 0 0.78 0407 0 0.48 392 05045 0 0.54 0410 0 0.32 395 0812 0 0.25 0413 0 0.31 394 6028 0 0.20 041® 0 0.45 395 0579 0 0.58 0432 0 9.57 396 0594 9 0.08 0437 0 0.40 397 9595 0 0.31 0445 0 0.81 39® 0491 0 0.46 O45S 0 0.19 399 0553 0 0.46 0459 0 0.42 400 0214 0 0.57 0461 0 0.23 481 0249 0 0.24 046® 0 0.34 402 0909 0 0.13 0469 0 0.35 403 0340 0 0.31 0470 0 0.05 404 0047 0 0.80 0 927 495 0277 0 0 139 0.7®

[0141] Identification of E. chaff eensis and E. canis immunodominant proteins. To further define and compare the antibody reactivity of these E. chajfeensis and E. canis proteins by ELISA, a panel of 8 HME and 8 CME sera were used. All patient and canine sera recognized E. chajfeensis or E. canis by IFA, respectively, with antibody titers ranging from 200 to 3200 (Figure 2). As previously described, well-defined immunodominant proteins (E. chajfeensis TRP120 or E. canis TRP19) were used as positive controls, respectively (Cardenas et al., 2007; Luo et al., 2010; Pritt and Dumler, 2019; Taques et al., 2020).

[0142] E. chajfeensis immunoreactive proteins (n = 40) were recognized by all or most of 8 HME sera. The top 7 proteins, including Ech_1061, 0725, 0350, 0506, 0679, 0494 and 0905, reacted strongly with all HME sera (similar to TRP120 positive control) and were considered immunodominant (ELISA ODeso values > 1.0) (Figure 2A). Additionally, some proteins (n = 33) reacted with all sera at lower levels (mean OD650= 0.5-1.0), and only reacted strongly with some HME sera. Thus, these immunoreactive proteins were considered subdominant. E. canis immunoreactive proteins (n = 15; ELISA ODeso values of > 0.5) were identified using 8 CME sera, but only 1 (Ecaj_0647) reacted strongly with most canine sera (mean ODeso value of > 1.0) and was considered immunodominant (Figure 2B). The positive control TRP19 reacted strongly with all CME sera. In addition, 3 E. canis proteins (Ecaj_0710, 0026 and 0274) with screening ODeso values of > 1.0 exhibited mean ODeso values of 0.5- 1.0 with 8 CME sera, and thus were considered subdominant.

[0143] Antibody epitopes of immunodominant proteins. The most immunoreactive proteins of E. chajfeensis and E. canis identified previously have conformation-dependent antibody epitopes (Luo et al., 2020; 2021). Thus, in this example, the conformation-dependence of 7 E. chajfeensis and 1 E. canis immunodominant proteins were also investigated by denaturing ELISA. After denaturation using urea, only 2 E. chajfeensis immunodominant protein (Ech_1061 and 0905) among top 7 still reacted weakly with 2 HME sera (mean ODeso < 0.2 from 8 sera), compared to the native IVTT proteins (mean OD650= 1.25 and 1.01, respectively). The remaining denatured E. chajfeensis proteins did not react with any HME patient sera, while the linear epitopecontaining major immunoreactive protein control (TRP120) was not affected by denaturation (Figure 3A) (McBride and Walker, 2010). These results indicate that these E. chajfeensis immunoreactive proteins have conformation-dependent antibody epitopes.

[0144] A synthetic peptide ELISA was also used to confirm the absence of major linear epitopes and presence of conformation-dependent epitopes in 2 selected E. chaffeensis immunodominant proteins (Ech_1061 and 0725) (Luo et al., 2008; Luo et al., 2009; McBride et al., 2011). Overlapping peptides (17-23 amino acids; 6 amino-acid overlap) covering each entire protein sequence were synthesized. The pooled HME sera used in the initial screening was used to test all peptides by ELISA (Figure 3B). None of these peptides reacted with HME sera, demonstrating that these E. chaffeensis immunodominant proteins do not contain major linear epitopes, consistent with ELISA results using native and denatured IVTT products (Figures 2A and 3A). The conformational dependence of epitopes was further examined in 7 new E. chaffeensis immunodominant proteins by dot immunoblot (Figure 3C). The immunoreactivity of native and denatured proteins was compared using an HME serum. After denaturation, these proteins did not react or reacted weakly with E. chaffeensis antibodies, consistent with the ELISA data in Figures 3 A and 3B. These results support the conclusion that the many immunodominant proteins of E. chaffeensis arc defined by conformation-dependent antibody epitopes.

[0145] The immunoreactivity of E. cams protein (Ecaj_0647) was only slightly reduced after denaturation by ELISA, indicating a major linear and minor conformation-dependent antibody epitopes were present. The well-defined E. canis major immunoreactive protein TRP19 containing a major linear antibody epitope was not affected by denaturation (Figure 4A). By dot immunoblot, denatured Ecaj_0647 protein reacted strongly with the E. canis antibodies, but at a lower level compared to native proteins, whereas TRP19 protein reacted at a level similar to the native proteins (Figure 4B). This result is consistent with the ELISA data in Figure 4A.

[0146] Analysis of antigenic proteins. To compile the antigenic repertories of E. chaffeensis and E. canis, data from this example was combined with recent studies and the results were summarized (FIG. 6). The combined data investigated 857 E. chaffeensis proteins and 817 E. canis proteins, excluding known antigens (such as TRPs and OMPs) and ribosomal proteins, and found 272 (32% of 857) and 112 (14% of 817) immunoreactive proteins, respectively. More importantly, many previously undefined immunodominant proteins in E. chaffeensis (n = 14) and E. canis (n = 18) were identified. In addition, numerous Ehrlichia subdominant proteins (n = 70 and n = 16, respectively) and other proteins exhibiting low immunoreactivity (n = 188 and n = 78, respectively) (FIG. 6) were identified. Figure 5 shows the quantity analysis of antigenic proteinsin E. chaffeensis and E. canis immunomes, including previously known antigens. Both Ehrlichia immunomcs contain ~3% immunodominant proteins; however, E. chaffeensis has more subdominant proteins and proteins exhibiting low immunoreactivity than E. canis (9% and 21% vs. 2% and 9%, respectively). Therefore, the proportion of the proteome that was not antigenic in E. canis and E. chaffeensis was 86% and 67%, respectively.

[0147] A comprehensive analysis of all new immunodominant proteins of E. chaffeensis and E. canis was performed. The immunoreactivity, E. canis orthologs and bioinformatic analysis of E. chaffeensis immunodominant proteins ranked by ELISA OD values are shown in Tables 1 and 2. All these proteins contain major conformational epitopes, and a majority of these proteins (n = 11) were small (< 250 amino acids) (Table 1). Among E. chaffeensis immunodominant proteins (n = 14), there were 6 hypothetical protein and 8 annotated proteins, including an outer membrane beta-barrel protein, a type IV secretion system component VirB3 and 6 enzymes involved in important biological processes, such as dehydrogenase, transferase, kinase, and phosphatase (Table 2). A bioinformatic analysis using multiple online prediction tools found that most E. chaffeensis immunodominant proteins (n = 8) were predicted to contain at least 1 (up to 8) transmembrane domain by TMHMM 2.0; however, using SignalP 6.0 and SecretomeP 2.0, only 3 proteins (Ech_0745, 0678 and 0679) were predicted to be secreted by a standard secretory signal peptide or a nonclassical (not signal peptide-dependent) protein secretion. Notably, most E. chaffeensis immunodominant proteins (n = 8) were identified as effectors by PREFFECTOR (Dhroso et al., 2018). Therefore, these proteins were also further analyzed to identify the type of secretion system substrates. Type I and type IV secretion systems (T1SS and T4SS) have been identified in Ehrlichia', however, a consensus sequence of type IV secretory motif R-X(7)-R-X-R- X-R (Vergunst et al., 2005) was not identified in any E. chaffeensis protein and none of the immunodominant proteins identified in this study were predicted to be type IV substrates by the S4TE 2.0 tool (Noroy et al., 2019). In contrast, a putative type I secretion signal (LDAVTSIF- enriched and KHPMWC-poor) (Delepelaire, 2004; Wakeel et al., 2011) was identified in the last 50 C-terminal residues of these proteins, suggesting that these proteins are type I secreted substrates. Ech_0875 protein showed the greatest difference between the residue occurrences of LDAVTSIF (72%) and KHPMWC (8%) in the last 50 C-terminal amino acids, whereas Ech_0745 showed the least difference (32% versus 18%). These results are consistent with previous reportsand support the conclusion that many of these E. chaffeensis immunodominant proteins are type I secreted effectors, although additional experimental validation is required (Table 2).TABLE 1 Tmmunoreactivity analysis of new E. ehaffeei-ws immunodominant proteins.Mean 27 cor / is orthologProtein , Conformational ANTIGENpro s . hklSA(Ecn_ tag „ epitope score Ecai tag ANTIGENproT,— - ‘ limmmoreacnvitv'' no. score1065 1.91 major 0.70 0857 0.75 -H-0578 1.30 major 0.80 * *1061* 1.25 major 0. H 0853 0.240875 1.20 major 0.06 0223 0 170725* 1.18 major 0.06 0339 0.101053 1.15 major 0.76 0846 0 580207 1.12 major 0.06 0796 0 040350s1.12 major 0.45 0659 0 440745 1.11 major 0.92 0324 0.920506* 1.10 major 0.15 * * »0678 1.07 major 0.54 0369 0.510679a1.03 major 0.13 0368 0.060494* 1.03 major 0.10 0534 0.10 +0905* 1.01 major 0.27 0200 0.23 a Mean OD6iCfrom 8 HME patient sera.4+, immunoreactive in immuiioscreenmg: not immunoreactive in mmiunoscreenins:immunodominant. *, E. amis ortholog not identified.* Proteins identified in this study. Others were identified in our recent studiesTABLE 2 Predicted feature* of new E. chaffeensis immunodcsninaatprotein*.No. ofProtein AAs / mass Transmembrane(Ech tag no.) Product (kDa) domain3-® Secretion T4SCEffector*1065 2-axGghitarate dehydrogenase E2 404'44 component0578 Hypothetical protein 185 / 21 - . . .106 V FMN 317 / 35 - - - 4 adeayiylti«tisferase;'dbofl3vin kinase0875 Phospliatidylglycersphosphatase 226 / 25 4A 0725* Methyttransferase domain- 264 / 29 containing protein1053 Hypothetical protein 193 / 22 4-0207 Hypothetical protein 176 / 10 + - - 40350* 2-ammo-4-hydroxy-6- 169 / 19 - . . 4 hydioxyinetiiyldihydi'opteridme- pyrophosphokinase0745 Hypothetical protein 118 / 13 - V - -0506* Hypothetical protein 96 / 11-0678 Hypothetical protein 239 / 25 4 +? - 40679sOuter membrane beta-barrel 283 / 26 4 - 4 protein0494sType IV secretion system protein 97 / 11 4 . . .”+, positive: negative.* Predicted by S4TE.“ PiecUcied by PREFFECTOR* Proteins identified in this study. Ottes were identified in our recent studies.■fPredicted by SecretomeP g Predicted by SignalP.

[0148] The analysis of E. canis immunodominant protein, ranked ELISA OD values, and comparison with E. chaffeensis orthologs are shown in Tables 3 and 4. The majority of these proteins (« = 14) contained major linear epitopes, including 5 proteins that also contained minor conformational epitopes. Four proteins (Ecaj_0128, 0348, 0857 and 0104) contained only major conformational epitopes (Table 3). A majority of E. canis immunodominant proteins (n = 11) were hypothetical, except for 7 annotated proteins including electron transport protein SCOl / SenC, an extracellular solute-binding protein, translation elongation factor 1A (EF-1A), 2-oxoglutarate dehydrogenase E2 component, peptidyl-prolyl cis-trans isomerase and 2 heat shock proteins (HSP60 and HSP70). Only 8 of these immunodominant proteins were smaller than 250 aminoacids. Bioinformatic analysis identified 7 E. canis proteins predicted to contain transmembrane domains and 5 proteins that were predicted to be secreted (all by nonclassical mechanism). Importantly, many of E. canis immunodominant proteins (n - 9) were identified as effectors by PREFFECTOR (Dhroso et al., 2018). Although 3 proteins (Ecaj_0126, 0259 and 0334) were predicted to be type IV substrates by the S4TE 2.0 tool, no type IV secretory signal (R-X[7]-R-X- R-X-R) was identified in any of these E. canis proteins. Moreover, a putative type I secretion signal (LDAVTSIF-enriched and KHPMWC-poor) in the C-terminus of these proteins suggested that most of these proteins are type I secreted substrates, consistent with a previous conclusion (Luo et al., 2020; 2021) (Table 4). Ecaj_0104 protein had the largest difference between the residue occurrences of LDAVTSIF (70%) and KHPMWC (6%) in the last 50 C-terminal amino acids, whereas the predicted type IV substrate Ech_0259 had the least difference (36% versus 24%).TABLE 3 lamiuiKsre^ivity analysis of new £. casts immimodommant proteins.. . .0126 2.13 no 0.96 0187 0 970717 1.35 sic 0.80 * *0151 1.72 nc 0 85 0976 0.77 -?012S 1.53 major 0.90 0189 0 8S 40636 1.52 no 0.76 0377 0.840073 1.51 no 0.89 0122 0 760920 1.44 nc 0.95 1148 0.85 -r020 1.44 no 0.61 * * *0259 1,27 mmor 0.93 OS25 0.92 4-0162 1.25 no 0.60 0960 0 430348 1.22 major 0.77 * » *0554 1.20 minor 0.84 0471 0 85 0857 1,19 major 0.75 1065 0.70 -H-0354 1 10 minor 0.87 0731 0.86 -t0647®' 1.08 minor 0.57 0365 0.58 -H-0104 1.02 major 0.39 9159 0.77 40737 1.00 miner 0.56* Mean ODj?? frcm CME pahent sera.A. immunoreactive m imtmffiosa«eaing; -, nor immunoreactive in immtmoscreening; -H-, immunodcmiHBnr, f L £. cfe$feea$jy onhciog not identified.® Proteins identified in this study. Others were identified in our recent studies.TABLE 4 Predicted features of iiew £.immunodominant proteins.No. of ammoPrarein acids / nms Transmembrane(Ecaj tag no / i Product (kDa> domain®* Secretion* T4S* Effected©919 Hypothetical protein 120 / 14 - 4 - 40126 Hypothetical protein 671 / 73 - 4 + 40717 Hypothetical protein 22 / 425 4 40151 Election transport protein 265 / 33 4 . . +SCOi / senC0128 Extracellular solute- 347 / 38 4 - binding protein, family 10636 Hypothetical protein 98 / 11 - -0073 Hype ills deal protein 92 / 10 - 4 - -0920 Hypothetical protein 182 / 20 - 4 - 40213 Hypothetical protein 328 / 36 4 . . .0259 Hypothetical protein 368 / 41 - 4 4 40162 Translation elongation 395 / 43 - . . .&ctor 1A(EF-1A / EF-Tu)0348 Hypothetical protein 535 / 59 4 -0554 Heat shock protein 634 / 69 - . . .HSP700857 2-exogiutarate 400 / 44 - . . . dehydrogenase E2 component0334 Peptkiyi-prolyl ds-trans 630 / 72 4 4 4 isomerase£>64T' chaperonin GroEL 552 / 61 - . . .(HSP50 family)0104 Hypothetical protein 182 / 20 4 . . .£>737 Hypothetical protein 194 / 21 - 4■’ Predicted by IMHMM. positive: -. negative.‘Predicted by SecretotneP. a' Predicted by S4TE' Predicted by PREFFECTOR.■■' Proteins idendSed in this sntdy. Others were identified in our recent studies.

[0149] The development of new and affordable biotechniques, such as next-generation genome sequencing, commercial gene synthesis and cloning, and in vitro protein expression, has made the analysis of entire bacterial immunomes feasible. Recent studies have established a rapid high-throughput antigen discovery strategy and this approach has been used to successfully identify many previously undiscovered immunoreactive proteins from E. chaffeensis and E. canis (Luo et al., 2020; 2021). In this investigation, many new immunodominant and subdominant ehrlichial proteins were identified, leading to the revelation of the antibody reactive antigenicrepertoires of E. chaffeensis and E. canis. This information will ultimately expand and accelerate vaccine and diagnostic development for the ehrlichioses.

[0150] The application of IVTT in antigen discovery is the key to identification of conformation dependent immunoreactive proteins, because IVTT generally expresses soluble proteins in native conformation, although posttranslational modifications may not exist (Shimizu et al., 2006; Carlson et al., 2012). The majority of bacterial B-cell epitopes are estimated to be conformational, and many pathogens are known to have immunoreactive proteins with conformational antibody epitopes (Portnyagina et al., 2018; Andrade etal., 2019; Liu etal., 2019; He et al., 2020). However, prior to recent studies, the defined E. chaffeensis and E. canis immunoreactive proteins were limited to those with only major linear epitopes due to the limitations in the approaches used for screening (McBride and Walker, 2010; Lina et al., 2016). Recent studies have revealed many immunoreactive proteins of E. chaffeensis and E. canis are predominated by conformation-dependent epitopes (Luo et al., 2020; 2021). Considering the antibody reactive proteins identified in recent studies, including the present examples, all of the new E. chaffeensis immunodominant proteins contain major conformation-dependent epitopes; however, linear antibody epitopes are predominant in E. canis immunodominant proteins, although many conformation-dependent epitopes in immunoreactive protein repertoires of E. canis were also identified.

[0151] Overall, E. canis immunodominant proteins appear to have higher ANTIGENpro score and rank than E. chaffeensis proteins, which may be related to the differences in the number of linear LGantibody epitopes found in E. canis antigenic proteins (Tables 1 and 3). All previously characterized major immunoreactive proteins of E. chaffeensis and E. canis that contain major linear epitopes, including TRPs, Ank200, OMPs and MSP4, arc represented in the top 250 list predicted by ANTIGENpro (Luo et al., 2020), suggesting that the machine learning model of ANTIGENpro may have a bias as it relates to known immunoprotective proteins used to train the algorithm that likely have a predominance of linear epitopes.

[0152] Many ehrlichial proteins that were previously considered to have unknown function (hypothetical), including TRPs and Anks, are now known to have defined functions during infection (Wakeel et al., 2009; Luo et al., 2011; Luo and McBride, 2012; Luo et al., 2018; Byerlyet al., 2021). Of proteins that make up the E. chaffeensis and E. canis proteomes, -25% are considered hypothetical or proteins with domain of unknown function (DUFs). Studies with other intracellular pathogens in Ehrlichia have determined that a large number of these proteins are immunoreactive (Cruz-Fisher et al., 2011; Liu et al., 2019). As a result, it has been recently reported that many immunoreactive proteins in the E. chaffeensis and E. canis proteomes are dominated by hypothetical proteins (Luo et al., 2020; 2021). An analysis of immunodominant proteins in Ehrlichia showed that while many hypothetical proteins are E. chaffeensis antigenic proteins, more immunoreactive hypothetical proteins exist in E. canis (Tables 2 and 4).

[0153] Among new immunodominant proteins identified in E. chaffeensis and E. canis in these studies, there were notable proteins with known functions. Interestingly, most E. chaffeensis proteins were predicted to be enzymes involved in important biological processes, such as energy production and conversion, coenzyme transport and metabolism, glycerophospholipid metabolism and protein regulation, demonstrating that these metabolically functional Ehrlichia proteins are also antigenic. Other more antigenically established proteins included an outer membrane betabarrel protein and a type IV secretion system protein VirB3 (Table 2). Multiple Ehrlichia! Anaplasma outer membrane proteins and type IV secretion system proteins, such as TRP, OMP, VirB and VirD, have also been previously identified as antigenic and protective (Sutten et al., 2010; Crocquet- Valdes et al., 2011; Kuriakose et al., 2012). Of new immunodominant proteins of E. canis identified in these studies, the antigenic annotated proteins are different from those identified in E. chaffeensis . For example, a peptidyl-prolyl cis-trans isomerase, an electron transport protein, an extracellular solute-binding protein, a translation elongation factor, and a heat shock protein HSP70 were identified in E. canis but not in E. chaffeensis (Table 4). Another heat shock protein GroEL (HSP60) has an ortholog in E. chaffeensis that was identified as the first immunodominant protein in 1993 (Sumner et al., 1993). Immunization with E. muris GroEL peptide is protective in a mouse model (Thomas et al., 2011). Notably, many immunodominant proteins of E. chaffeensis and E. canis were predicted to contain transmembrane domains, further highlighting this feature in many antigenic proteins.

[0154] Unlike the previously identified immunoreactive protein orthologs of Ehrlichia and E. canis that contain major linear epitopes, such as TRPs, Anks and OMPs, only 2 respective E. canis or E. chaffeensis orthologs (2-oxoglutarate dehydrogenase E2 component and GroEL) inthese studies were also found to be immunodominant, and only a minority of E. chaffeensis or E. canis orthologs shared immunorcactivity, although the respective orthologs were identified for the majority of Ehrlichia immunodominant proteins (Tables 1 and 3). These findings suggest that E. chaffeensis and E. canis do not have similar orthologous antigenic proteins as might be expected, and the antibody epitopes in majority of Ehrlichia immunodominant proteins are not conserved between E. chaffeensis and E. canis. Notably, E. chaffeensis immunodominant proteins reacted with HME sera more consistently than E. canis proteins with CME sera (Figure 2). Hence, it is possible that E. canis immunoreactive proteins are more antigenically variable among different E. canis strains. Antigenic diversity in E. canis is well defined, including TRP36, a major immunoreactive protein, and extensive phylogenetic analysis of TRP36 genes has identified several E. canis genogroups in North America, Central America, South America, Africa, Europe and Asia, which have antigenic variability (Zhang et al., 2008; Aguiar et al., 2013; Arroyave et al., 2020). Therefore, antigenic variability in some E. canis proteins may also contribute to the lower ratio of immunoreactive proteins found in E. canis compared to E. chaffeensis in this study and the entire immunomes. In addition, the average size of new E. chaffeensis immunodominant proteins appear to be smaller than that of E. canis. A vast majority of 14 new E. chaffeensis immunodominant proteins (n = 11) are small (< 250 amino acids), while only 8 of 18 E. canis proteins are small (Tables 2 and 4). Collectively, these results indicate the fundamental differences in antigenic protein profiles between E. chaffeensis and E. canis, which is potentially valuable information for development of specific diagnostics and vaccines for these Ehrlichia species.

[0155] Consistent with recent reports (Luo et al., 2020; 2021), the majority of E. chaffeensis and E. canis immunodominant proteins were predicted to be type I secreted effectors, despite that fact that only 8 proteins were predicted to be secreted by SignalP or SecretomeP. This reinforces the conclusion that in addition to previously defined major immunoreactive proteins that have linear epitopes, such as TRPs and Ank200, there are also other proteins with conformational epitopes that appeal' to be T1SS substrates. These results further support an important role of the T1SS in Ehrlichia infection and potentially immunity (Wakeel et al., 2011). It has been shown that several ehrlichial T1S substrates (TRPs) play important roles in pathobiology by regulating important cellular processes to promote ehrlichial survival (Dunphy et al., 2013; Lina et al., 2016; Bui et al., 2023).

[0156] Collectively, this investigation and combined with other recent studies, has successfully defined the antigenic proteins contained in the E. chaffeensis and E. canis protcomcs. It is expected that this information will provide a defined set of antigens from which a rational vaccine and diagnostic development strategy can be implemented and tested for HME and CME. Further studies are also needed to determine the T-cell epitopes, secretion mechanism and roles of these proteins in ehrlichial pathobiology and immunity.Example 2 - Materials and Methods

[0157] The following materials and methods were used to generate the results provided in Example 1.

[0158] Gene synthesis and cloning. E. chaffeensis (Arkansas strain) and E. canis (Jake strain) gene sequences are available in the Integrated Microbial Genomes (IMG) (img.jgi.doe.gov) (Chen et al., 2019) and GenBank (ncbi.nlm.nih.gov / genbank). Ehrlichia genes were codon- optimized, chemically synthesized and cloned into pIVEX2.3d vector (containing a 6xHis-tag sequence) by Twist Bioscience (San Francisco, CA) or GenScript (Piscataway, NJ). Plasmids were transformed into Escherichia coli to amplify, then extracted and lyophilized by the manufacturer.

[0159] In vitro transcription and translation (IVTT). In vitro expression of Ehrlichia proteins was performed using the NEBExprcss cell-free E. coli protein synthesis system (New England Biolabs, Ipswich, MA). Lyophilized plasmids were reconstituted in water and purified using the UltraClean 96 PCR cleanup kit (Qiagen, Germantown, MD). Plasmids were then added to E. coli extract and a reaction premix in a 96- well plate and incubated at 37 °C for 3 h with orbital shaking (300 rpm) according to the manufacturer’s instructions.

[0160] HME and CME antisera. HME patient sera from the Centers for Disease Control and Prevention (Atlanta, GA), Vanderbilt University School of Medicine (Nashville, TN), Washington University and the St. Louis Children’s Hospital (St. Louis, MO) was utilized. CME sera were obtained from naturally infected dogs from the United States and Colombia. To avoid reactions with non-specific polyreactive IgM antibodies, which have been previously described in humans (Jones et al., 2012), assays were performed with convalescent sera and bound antibody detected with anti-IgG (H+L) secondary antibodies.

[0161] Dot immunoblot. The expression of Ehrlichia proteins by IVTT was confirmed by dot immunoblot with horseradish peroxidase (HRP)-labclcd mouse anti-His tag monoclonal antibody (1:500; GenScript) as described previously (Luo et al., 2020). The immunoreactivity of native and denatured proteins was also examined by dot immunoblot using IVTT-expressed proteins purified by MagneHis protein purification system (Promega, Madison, WI) according to the manufacturer. Immunoblots were probed with either HME or CME serum (1:200) and developed with TMB 1-component substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD).

[0162] ELISA immunoscreening. The immunoreactivity of Ehrlichia IVTT-expressed proteins was performed by capturing His-tagged IVTT proteins on an ELISA plate coated with anti-His tag antibody as previously described with minor modifications (Luo et al., 2020). ELISA was performed with HME or CME sera (1:200) and bound antibody detected with alkaline phosphatase-labeled rabbit anti-human IgG (H+L) (1:7,000; Abeam, Cambridge, MA) or anti-dog IgG (H+L) secondary antibodies (1:5000) and BluePhos substrate (Kirkegaard & Perry Laboratories). Dilution buffer containing 4 M urea was used to denature IVTT-expressed proteins and the diluted protein was incubated for 10 min at 99°C before cooling on ice and coating the plate (or membrane for dot blot). Optical density was measured at 650 nm (ODeso) on a SpectraMax iD5 plate reader (Molecular Devices, Sunnyvale, CA) and ODeso values represent the mean reading from 3 wells (± standard deviation) after negative control (IVTT negative protein control and a normal human or canine serum control) background subtraction. A sample ELISA OD650 value of > 0.2 was considered positive and > 0.5 a strong positive after subtracting the negative control ODeso reading (background). The proteins with mean ELISA ODesoof > 1.0 from multiple sera were considered immunodominant and proteins with mean ELISA ODeso of 0.5-1.0 subdominant.

[0163] Peptide ELISA. To identify linear antibody epitopes, ELISAs were performed using overlapping peptides (17-23 amino acids; 6 amino acid overlap) (Luo et al., 2009). All peptides were commercially synthesized and supplied as a lyophilized powder (GenScript) and resuspended in molecular biology grade water (1 mg / ml). A small amount of NH4OH, acetic acid or dimethyl sulfone was added to help dissolve some acidic, basic, or hydrophobic peptides, respectively, according to peptide solubility guidelines from the manufacturer.

[0164] Indirect fluorescent-antibody assay (IFA). The antibody titers in sera from HME patients and CME dogs were determined by IFA as previously described (Luo et al., 2020). Antigen slides were prepared from E. chaffeensis (Arkansas)-infected THP-1 cells or E. cams (Jake)-infected DH82 cells. Slides were examined with a BX61 epifluorescence microscope (Olympus, Japan).

[0165] Bioinformatic analysis. Online bioinformatic prediction tools used in this study include ANTIGENpro (scratch.proteomics.ics.uci.edu), TMHMM 2.0 (services.healthtech.dtu.dk / service.php?TMHMM-2.0), SignalP 6.0(services. healthtech.dtu.dk / service.php7SignalP-6.0), SecretomeP 2.0(services.healthtech.dtu.dk / service. php?SecretomeP-2.0), S4TE 2.0 (sate.cirad.fr), and PREFFECTOR (draco .cs . wpi.edu / preffector) .* * *

[0166] All of the 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 methods 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. 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Claims

WHAT IS CLAIMED IS:

1. A pharmaceutical composition comprising a nucleic acid comprising an open reading frame encoding Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NO:2), Ech_0350 (SEQ ID NO:3), Ech 0506 (SEQ ID NO:4), Ech 0679 (SEQ ID NO:5), Ech 0494 (SEQ ID NO:6), Ech_0905 (SEQ ID NO:8), Ecaj_0647 (SEQ ID NO:7), or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto; wherein the composition is formulated in a lipid nanoparticle or a viral vector.

2. The pharmaceutical composition of claim 2, wherein the nucleic acid is a ribonucleic acid (RNA).

3. The pharmaceutical composition of claim 2, wherein the nucleic acid is an mRNA further comprising a 5' untranslated region (UTR) and a 3' UTR.

4. The pharmaceutical composition of claim 3, wherein the mRNA comprises at least one analogue of a naturally occurring nucleotide or wherein the mRNA is chemically modified.

5. The pharmaceutical composition of claim 4, wherein the analogue is selected from the group consisting of phosphorothioates, phosphoramidates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, and inosine.

6. The pharmaceutical composition of claim 4 wherein the mRNA comprises pseudouridine, a 5' cap analog, or a poly(A) tail.

7. The pharmaceutical composition of claim 6, wherein the 5' cap analog is 7mG(5')ppp(5')NlmpNp.

8. The pharmaceutical composition of claim 6, wherein the chemical modification is a 1- methylpseudouridine modification or a 1 -ethylpseudouridine modification.

9. The pharmaceutical composition of any one of claims 2-8, wherein the mRNA comprises a 5' untranslated region (UTR) and a 3' UTR.

10. The pharmaceutical composition of any one of claims 3-9, wherein the mRNA is comprised in liposomes, lipid nanoparticlcs, or a viral vector.

11. The pharmaceutical composition of claim 10, wherein the liposomes or lipid nanoparticles comprise an ionizable cationic lipid, a neutral lipid (e.g., DSPC), sterol (e.g.. cholesterol), and / or a PEG-modified lipid (e.g., PEG-DMG or PEG-DMA).

12. The pharmaceutical composition of any one of claims 2-11, wherein the RNA encodes the polypeptide for secretion.

13. The pharmaceutical composition of any one of claims 2-11, wherein the RNA encodes the polypeptide as an intracellular protein.

14. The pharmaceutical composition of any one of claims 2-11, wherein the polypeptide is comprised in a fusion protein, preferably wherein the fusion protein comprises a transmembrane region.

15. The pharmaceutical composition of claim 1, wherein the nucleic acid is a DNA.

16. The pharmaceutical composition of claim 15, wherein the DNA is comprised in a viral vector.

17. The pharmaceutical composition of claim 15, wherein the viral vector is an adenovirus, or an adeno-associated virus (AAV).

18. The pharmaceutical composition of any one of claims 1-17, wherein the pharmaceutical composition is further characterized as an immunogenic composition, an immunoreactive composition, and / or a vaccine composition.

19. A pharmaceutical composition comprising Ech_1061 (SEQ ID NO: 1), Ech_0725 (SEQ ID NO:2), Ech_0350 (SEQ ID NO:3), Ech_0506 (SEQ ID NO:4), Ech_0679 (SEQ ID NO:5), Ech_0494 (SEQ ID NO:6), Ech_0905 (SEQ ID NO:8), Ecaj_0647 (SEQ ID NO:7), or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, and an excipient.

20. The pharmaceutical composition of claim 19, wherein the composition further comprises an adjuvant.

21. The pharmaceutical composition of claim 20, wherein the adjuvant comprises a triterpenoid saponin (preferably Quil A), a sterol, and / or an immunostimulatory oligonucleotide (preferably a CpG-containing ODN).

22. The pharmaceutical composition of claim 21, wherein the CpG-containing ODN is 5’ JU*C-G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C* G*T 3' (SEQ ID NO: 9), wherein refers to a phosphorothioate bond,refers to a phosphodiester bond, and “JU” refers to 5'-Iodo-2'-deoxyuridine.

23. The pharmaceutical composition of any one of claims 19-22, wherein the composition comprises an E. cams bacterin or an E. chaffeensis bacterin; preferably wherein the E. canis bacterin or the E. chaffeensis bacterin is a heat-inactivated or chemically-inactivated bacterin, preferably wherein the chemically-inactivated bacterin was inactivated with formaldehyde, formalin, bi-ethylene amine, radiation, ultraviolet light, beta-propiolactone treatment, or formaldehyde.

24. The pharmaceutical composition of any one of claims 19-23, wherein the Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NOG), Ech_0350 (SEQ ID NOG), Ech_0506 (SEQ ID NOG), Ech_0679 (SEQ ID NOG), Ech_0494 (SEQ ID NOG), Ech_0905 (SEQ ID NOG), Ecaj_0647 (SEQ ID NOG), or the polypeptide is comprised in a multimer or fusion protein.

25. The pharmaceutical composition of any one of claims 19-24, wherein the pharmaceutical composition is further characterized as an immunogenic composition, an immunoreactive composition, and / or a vaccine composition.

26. A method of detecting antibodies that specifically bind an Ehrlichia organism in a test sample, comprising:(a) contacting a polypeptide comprising or consisting of a sequence of Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NOG), Ech_0350 (SEQ ID NOG), Ech_0506 (SEQ ID NOG), Ech_0679 (SEQ ID NOG), Ech_0494 (SEQ ID NOG), Ech_0905 (SEQ ID NOG), Ecaj_0647 (SEQ ID NOG), or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, with the test sample, underconditions 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.

27. The method of claim 26, wherein the Ehrlichia organism is an Ehrlichia chaffeensis organism.

28. The method of any one of claims 26-27, wherein the Ehrlichia organism is an Ehrlichia canis organism.

29. The method of any one of claims 26-28, wherein the step of detecting comprises performing an enzyme-linked immunoassay, a radioimmunoassay, an immunoprecipitation, a fluorescence immunoassay, a chemiluminescent assay, an immunoblot assay, a lateral flow assay, a flow cytometry assay, a multiplex immunoassay, a mass spectrometry assay, or a particulate-based assay.

30. The method of claim 29, wherein the step of detecting comprises a lateral flow assay or an enzyme-linked immunoassay, wherein the enzyme-linked immunoassay is an ELISA.

31. The method of any one of claims 26-30, wherein the polypeptide is further characterized as an isolated polypeptide.

32. A method of identifying an Ehrlichia infection in a mammalian subject comprising: (a) contacting a biological sample from the subject with an polypeptide comprising or consisting of Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NO:2), Ech_O35O (SEQ ID NO:3), Ech_0506 (SEQ ID NO:4), Ech_0679 (SEQ ID NO:5), Ech_0494 (SEQ ID NO:6), Ech_0905 (SEQ ID NO:8), Ecaj_0647 (SEQ ID NO:7), or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, 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.

33. The method of claim 32, wherein the step of detecting comprises performing an enzyme- linked immunoassay, a radioimmunoassay, an immunoprecipitation, a fluorescence immunoassay, a chemiluminescent assay, an immunoblot assay, a lateral flow assay, a flow cytometry assay, a multiplex immunoassay, a dipstick test, or a particulate-based assay.

34. The method of claim 32, wherein the subject is a human.

35. The method of claim 32, wherein the subject is a dog.

36. The method of any one of claims 32-35, wherein the polypeptide is further characterized as an isolated polypeptide.

37. A polypeptide comprising or consisting of Ech_1061 (SEQ ID NO: 1), Ech_0725 (SEQ ID NO:2), Ech_0350 (SEQ ID NO:3), Ech_0506 (SEQ ID NO:4), Ech_0679 (SEQ ID NO:5), Ech_0494 (SEQ ID NO:6), and Ecaj_0647 (SEQ ID NO:7), Ech_0905 (SEQ ID NO:8), Ecaj_0647 (SEQ ID NO:7), or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, wherein the polypeptide is immobilized on a surface of a support substrate.

38. The polypeptide of claim 37, wherein the support substrate comprises latex, polystyrene, nylon, nitrocellulose, cellulose, silica, agarose, or magnetic resin.

39. The polypeptide of any one of claims 37-38, 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.

40. The polypeptide of any one of claims 37-39, wherein the polypeptide is comprised in a kit.

41. The polypeptide of any one of claims 37-39, wherein the polypeptide is produced via peptide synthesis or in vitro transcription and translation (IVTT).

42. The polypeptide of any one of claims 37-39, wherein the polypeptide is recombinantly produced.

43. The method of any one of claims 37-42, wherein the polypeptide is further characterized as an isolated polypeptide.

44. A polypeptide comprising or consisting of Ech_1061 (SEQ ID NO: 1), Ech_0725 (SEQ ID NO:2), Ech_0350 (SEQ ID NO:3), Ech_0506 (SEQ ID NO:4), Ech_0679 (SEQ ID NO:5), Ech_0494 (SEQ ID NO:6), Ech 0905 (SEQ ID NO:8), Ecaj_0647 (SEQ ID NO:7), or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, wherein the polypeptide is covalently attached to or bound to a detectable label.

45. The polypeptide of claim 44, wherein the detectable label is a fluorescent label, a radioactive label, an enzyme label, or a luminescent nanoparticle.

46. The polypeptide of claim 45, wherein the luminescent nanoparticle is a luminescent rare earth nanoparticle, a luminous nanoparticle, or a strontium aluminate nanoparticle.

47. The polypeptide of any one of claims 44-46, wherein the polypeptide is comprised in a kit.

48. The polypeptide of any one of claims 44-47, wherein the polypeptide is produced via peptide synthesis or in vitro transcription and translation (IVTT).

49. The polypeptide of any one of claims 44-47, wherein the polypeptide is rccombinantly produced.

50. The method of any one of claims 44-49, wherein the polypeptide is further characterized as an isolated polypeptide.

51. A kit comprising: (a) the polypeptide of any one of claims 44-50, (b) an anti-dog or antihuman secondary antibody linked to a reporter molecule; and, (c) an appropriate reagent for detection of the reporter molecule.

52. The kit of claim 51 , wherein the polypeptide is immobilized on a membrane or a microtiter plate.

53. The kit of any one of claims 51-52, wherein the reporter molecule is selected from the group consisting of luciferase, horseradish peroxidase, a luminous nanoparticlc, P- galactosidase, and a fluorescent label.

54. The kit of claim 53, wherein the luminous nanoparticle is a strontium aluminate nanoparticle.

55. The kit of claim of any one of claims 51-54, wherein the kit further comprises a dilution buffer for dog or human serum.

56. The kit of claim of any one of claims 51-55, wherein the kit comprises a lateral flow immunoassay or a lateral flow immunochromatographic assay.

57. The kit of claim of any one of claims 51-56, wherein the kit comprises an enzyme-linked immunosorbent assay (ELISA).

58. A method of inducing an immune response in a mammalian subject comprising administering to the subject an effective amount of a pharmaceutical composition comprising a polypeptide comprising or consisting of Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NO:2), Ech_0350 (SEQ ID NO:3), Ech_0506 (SEQ ID NO:4), Ech_0679 (SEQ ID NO:5), Ech_0494 (SEQ ID NO:6), and Ecaj_0647 (SEQ ID NO:7), Ech_0905 (SEQ ID NO:8), Ecaj_0647 (SEQ ID NO:7), or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, or a nucleic acid encoding a polypeptide sequence of Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NO:2), Ech 0350 (SEQ ID NO:3), Ech 0506 (SEQ ID NO:4), Ech 0679 (SEQ ID NO:5), Ech_0494 (SEQ ID NO:6), Ech_0905 (SEQ ID NO:8), Ecaj_0647 (SEQ ID NO:7), or encoding a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto.

59. The method of claim 58, wherein the nucleic acid is an mRNA.

60. The method of claim 59, wherein the mRNA comprises at least one analogue of a naturally occurring nucleotide or wherein the mRNA is chemically modified.

61. The method of claim 60, wherein the analogue is selected from the group consisting of phosphorothioatcs, phosphoramidatcs, peptide nucleotides, mcthylphosphonatcs, 7- deazaguanosine, 5-methylcytosine, and inosine.

62. The method of claim 60 wherein the mRNA comprises pseudouridine, a 5' cap analog, or a poly( A) tail.

63. The method of claim 62, wherein the chemical modification is a 1 -methylpseudouridine modification or a 1 -ethylpseudouridine modification.

64. The method of claim 62, wherein the mRNA comprises a 5' untranslated region (UTR) and a 3' UTR.

65. The method of any one of claims 59-64, wherein the mRNA is comprised in liposomes, lipid nanoparticles, preferably wherein the liposomes or lipid nanoparticles comprise an ionizable cationic lipid, a neutral lipid (e.g. , DSPC), sterol (e.g. , cholesterol), and / or a PEG- modifted lipid (e.g., PEG-DMG or PEG-DMA).

66. The method of any one of claims 58-65, wherein the nucleic acid is a DNA.

67. The method of claim 66, wherein the DNA is comprised in a viral vector.

68. The method of claim 67, wherein the viral vector is an adenovirus, or an adeno-associated virus (AAV).

69. The method of claim 58, wherein the method comprises administering the pharmaceutical composition of any one of claims 1-25 to the mammalian subject.

70. The method of any one of claims 58-69, wherein the subject is a human or a dog.

71. The method of any one of claims 58-70, wherein the pharmaceutical composition is administered subcutaneously, intramuscularly, nasally, via inhalation or aerosol delivery, or intradermally.

72. The method of any one of claims 58 or 70-71, wherein the polypeptide comprises or consists of Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NO:2), Ech_0350 (SEQ IDN0:3), Ech_0506 (SEQ ID N0:4), Ech_0679 (SEQ ID N0:5), Ech_0494 (SEQ ID N0:6), Ecaj_0647 (SEQ ID N0:7), Ech_0905 (SEQ ID N0:8), or Ecaj_0647 (SEQ ID NO:7).

73. The method of any one of claims 58-72, wherein the method further comprises administering an Ehrlichia bacterin or an adjuvant to the mammalian subject.

74. The method of any one of claims 58-73, wherein the pharmaceutical composition is further characterized as an immunogenic composition, an immunoreactive composition, and / or a vaccine composition.

75. A method of treating an Ehrlichia chaff eensis infection in a subject comprising:(a) contacting a biological sample from the subject with an isolated polypeptide comprising or consisting of a sequence of Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NO:2), Ech_0350 (SEQ ID NO:3), Ech_0506 (SEQ ID NO:4), Ech_0679 (SEQ ID NO:5), Ech_0494 (SEQ ID NO:6), and Ecaj_0647 (SEQ ID NO:7), Ech_0905 (SEQ ID NO:8), Ecaj_0647 (SEQ ID NO:7), or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, 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 the subject has an Ehrlichia chaffeensis infection; and(c) administering a therapeutic compound to treat Ehrlichia infection in the subject.

76. The method of any one of claims 75, wherein the step of detecting comprises performing an enzyme-linked immunoassay, a radioimmunoassay, an immunoprecipitation, a fluorescence immunoassay, a chemiluminescent assay, an immunoblot assay, a lateral flow assay, a flow cytometry assay, a multiplex immunoassay, a dipstick test, or a particulatebased assay.

77. The method of claim 75, wherein the subject is a dog or a human.

78. The method of any one of claims 75-77, wherein the therapeutic compound is an antibiotic.

79. The method of claim 78, wherein the antibiotic is doxycycline.

80. The method of any one of claims 75-77, wherein the therapeutic compound is an antibody, preferably wherein the antibody is a polyclonal antibody, a monoclonal antibody, a mammalian antibody, or a humanized antibody.

81. The method of any one of claims 75-80, wherein the antibody is present in a multimer.

82. An in vitro method of detecting an ehrlichiosis infection in a mammalian subject, comprising:(a) obtaining a biological sample from the mammalian subject, wherein the biological sample is preferably serum or blood; and(b) performing a polymerase chain reaction (PCR) amplification that can selectively expand a nucleic acid encoding Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NO:2), Ech_0350 (SEQ ID NO:3), Ech_0506 (SEQ ID NO:4), Ech_0679 (SEQ ID NO:5), Ech_0494 (SEQ ID NO:6), Ech_0905 (SEQ ID NO:8), Ecaj_0647 (SEQ ID NO:7), or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto; wherein expansion of the nucleic acid indicates that the mammalian subject has ehrlichiosis.

83. The pharmaceutical composition of any one of claims 1 to 25 for use in preventing, in prophylaxis of, and / or treating ehrlichiosis or an infection by E. chaffeensis or E. canis in a subject.

84. A nucleic acid as defined in any one of claims 1 to 18 for use in preventing and / or treating ehrlichiosis or an Ehrlichia chaffeensis infection in a subject.

85. A polypeptide for use in preventing and / or treating ehrlichiosis or an Ehrlichia chaffeensis infection in a subject, wherein said polypeptide is Ech_1061 (SEQ ID NO:1), Ech_0725 (SEQ ID NO:2), Ech_O35O (SEQ ID NO:3), Ech_0506 (SEQ ID NO:4), Ech_0679 (SEQ ID NO:5), Ech_0494 (SEQ ID NO:6), Ech_0905 (SEQ ID NO:8), Ecaj_0647 (SEQ IDN0:7), or a polypeptide having at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto.

86. The pharmaceutical composition for use according to claim 83, the nucleic acid for use according to claim 84 or the polypeptide for use according to claim 85, wherein said pharmaceutical composition, nucleic acid or polypeptide induces an immune response in the subject upon administration to the subject.

87. The pharmaceutical composition of claim 86, wherein said immune response is protective for the subject from ehrlichiosis or an infection by E. chaff eensis or E. canis.

88. The pharmaceutical composition for use according to claim 83 or 86, the nucleic acid for use according to claim 84 or 86 or the polypeptide for use according to claim 85 or 86, wherein said pharmaceutical composition, nucleic acid or polypeptide is to be administered subcutaneously, intramuscularly, nasally, via inhalation or aerosol delivery, or intradermally to the subject.

89. The pharmaceutical composition for use according to any one of claims 83, 86 and 88, the nucleic acid for use according to any one of claims 84, 86 and 88, or the polypeptide for use according to any one of claims 85 to 88, wherein said pharmaceutical composition, nucleic acid or polypeptide is to be administered to the subject in combination with an Ehrlichia bacterin or an adjuvant.

90. The pharmaceutical composition of claim 89, wherein the pharmaceutical composition results in an immune response, preferably a protective immune response, for the subject against ehrlichiosis or infection by E. chaffeensis or E. canis.

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