AU762315B2 - Homologous 28-kilodalton immunodominant protein genes of ehrlichia canis and uses thereof - Google Patents

Description

WO 00/32745 PCT/[LS99/28075 HOMOLOGOUS 28-KILODALTON IMMUNODOMINANT

PROTEIN

GENES OF EHRLICHIA CANIS AND USES THEREOF BACKGROUND OF THE INVENTION Field of the Invention The present invention relates generally to the field of molecular biology. More specifically, the present invention relates to molecular cloning and characterization of homologous 28-kDa protein genes in Ehrlichia canis and a multigene locus encoding the 28-kDa homologous proteins of Ehrlichia canis and uses thereof.

Description of the Related Art Canine ehrlichiosis, also known as canine tropical pancytopenia, is a tick-borne rickettsial disease of dogs first described in Africa in 1935 and the United States in 1963 (Donatien and Lestoquard, 1935; Ewing, 1963). The disease became better recognized after an epizootic outbreak occurred in United States military dogs during the Vietnam War (Walker et al., 1970) The etiologic agent of canine ehrlichiosis is Ehrlichia canis, a small, gram-negative, obligate intracellular bacterium which exhibits tropism for mononuclear phagocytes (Nyindo et al., 1971) and is transmitted by the brown dog tick, Rhipicephalus sanguineus (Groves et al., 1975). The progression of canine ehrlichiosis occurs in three phases, acute, subclinical and chronic. The acute phase is characterized by fever, anorexia, depression, lymphadenopathy and mild thrombocytopenia (Troy and Forrester, 1990). Dogs typically WO 00/32745 PCT/US99/28075 recover from the acute phase, but become persistently infected carriers of the organism without clinical signs of disease for months or even years (Harrus et al., 1998). A chronic phase develops in some cases that is characterized by thrombocytopenia, hyperglobulinemia, anorexia, emaciation, and hemorrhage, particularly epistaxis, followed by death (Troy and Forrester, 1990).

Molecular taxonomic analysis based on the 16S rRNA gene has determined that E. canis and E. chaffeensis, the etiologic agent of human monocytic ehrlichiosis (HME), are closely related (Anderson et al., 1991; Anderson et al., 1992; Dawson et al., 1991; Chen et al., 1994). Considerable cross reactivity of the 64, 47, 40, 30, 29 and 23kDa antigens between E. canis and E. chaffeensis has been reported (Chen et al., 1994; Chen et al., 1997; Rikihisa et al., 1994; Rikihisa et al., 1992). Analysis of immunoreactive antigens with human and canine convalescent phase sera by immunoblot has resulted in the identification of numerous immunodominant proteins of E. canis, including a 30-kDa protein (Chen et al., 1997). In addition, a protein of E. canis has been described as a major immunodominant antigen recognized early in the immune response that is antigenically distinct from the 30-kDa protein of E. chaffeensis (Rikihisa et al., 1992; Rikihisa et al., 1994). Other immunodominant proteins of E canis with molecular masses ranging from 20 to 30-kDa have also been identified (Brouqui et al., 1992; Nyindo et al., 1991; Chen et al., 1994; Chen et al., 1997).

Recently, cloning and sequencing of a multigene family (omp-1) encoding proteins of 23 to 28-kDa have been described for E chaffeensis (Ohashi et al., 1998). The 28-kDa immunodominant outer membrane protein gene (p28) of E. chaffeensis, homologous to the Cowdria rumninantium map-1 gene, was cloned. Mice immunized with recombinant P28 were protected against challenge infection with the WO 00/32745 PCT/US99/28075 homologous strain according to PCR analysis of periperal blood 5 days after challenge (Ohashi et al., 1998). Molecular cloning of two similar, but nonidentical, tandemly arranged 28-kDa genes of E. canis homologous to E. chaffeensis omp-1 gene family and C. rumanintium map-1 gene has also been reported (Reddy et al., 1998).

The prior art is deficient in the lack of cloning and characterization of new homologous 28-kDa immunoreactive protein genes of Ehrlichia canis and a single multigene locus containing the homologous 28-kDa protein genes. Further, The prior art is deficient in the lack of recombinant proteins of such immunoreactive genes of Ehrlichia canis. The present invention fulfills this long-standing need and desire in the art.

SUMMARY OF THE INVENTION The present invention describes the molecular cloning, sequencing, characterization, and expression of homologous mature 28-kDa immunoreactive protein genes of Ehrlichia canis (designated Eca28-1, ECa28SA3 and ECa28SA2), and the identification of a single locus (5.592-kb) containing five 28-kDa protein genes of Ehrlichia canis (ECa28SAI, ECa28SA2, ECa28SA3, Eca28-1 and ECa28-2).

Comparison with E. chaffeensis and among E. canis 28-kDa protein genes revealed that ECa28-1 shares the most amino acid homology with the E. chaffeensis omp-1 multigene family and is highly conserved among E. canis isolates. The five 28-kDa proteins were predicted to have signal peptides resulting in mature proteins, and had amino acid homology ranging from 51 to 72%. Analysis of intergenic regions revealed hypothetical promoter regions for each gene, suggesting that these genes may be independently and differentially expressed.

Intergenic noncoding regions ranged in size from 299 to 355-bp, and WO 00/32745 PCT/US99/28075 were 48 to 71% homologous.

In one embodiment of the present invention, there are provided DNA sequences encoding a 30-kDa immunoreactive protein of Ehrlichia canis. Preferably, the protein has an amino acid sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4 and SEQ ID No. 6, and the gene has a nucleic acid sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3 and SEQ ID No. and is a member of a polymorphic multiple gene family. Generally, the protein has an N-terminal signal sequence which is cleaved after post-translational process resulting in the production of a mature 28kDa protein. Still preferably, the DNAs encoding 28-kDa proteins are contained in a single multigene locus, which has the size of 5.592 kb and encodes all five homologous 28-kDa proteins of Ehrlichia canis.

In another embodiment of the present invention, there is provided an expression vector comprising a gene encoding a 28-kDa immunoreactive protein of Ehrlichia canis and capable of expressing the gene when the vector is introduced into a cell.

In still another embodiment of the present invention, there is provided a recombinant protein comprising an amino acid sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4 and SEQ ID No. 6. Preferably, the amino acid sequence is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3 and SEQ ID No. 5. Preferably, the recombinant protein comprises four variable regions which are surface exposed, hydrophilic and antigenic. The recombinant protein may be useful as an antigen.

In yet another embodiment of the present invention, there is provided a method of producing the recombinant protein, comprising the steps of obtaining a vector that comprises an expression region comprising a sequence encoding the amino acid WO 00/32745 PCT[US99/28075 sequence selected from the group consisting of SEQ ID No. 2. SEQ ID No. 4 and SEQ ID No. 6 operatively linked to a promoter; transfecting the vector into a cell; and culturing the cell under conditions effective for expression of the expression region.

The invention may also be described in certain embodiments as a method of inhibiting Ehrlichia canis infection in a subject comprising the steps of: identifying a subject suspected of being exposed to or infected with Ehrlichia canis; and administering a composition comprising a 28-kDa antigen of Ehrlichia canis in an amount effective to inhibit an Ehrlichia canis infection. The inhibition may occur through any means such as, i.e. the stimulation of the subject's humoral or cellular immune responses, or by other means such as inhibiting the normal function of the 28-kDa antigen, or even competing with the antigen for interaction with some agent in the subject's body.

Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions of the invention briefly summarized above may be had by reference to certain embodiments thereof which are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted, however, that the appended WO 00/32745 PCT/US99/28075 drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope.

Figure 1 shows nucleic acid sequence (SEQ ID No. 1) and deduced amino acid sequence (SEQ ID No. 2) of ECa28-1 gene including adjacent 5' and 3' non-coding sequences. The ATG start codon and TAA termination are shown in bold, and the 23 amino acid leader signal sequence is underlined.

Figure 2 shows SDS-PAGE of expressed recombinant ECa28-1-thioredoxin fusion protein (Lane 1, arrow) and 16-kDa thioredoxin control (Lane 2, arrow), and corresponding immunoblot of recombinant ECa28-l-thioredoxin fusion protein recognized by covalescent-phase E. canis canine antiserum (Lane 3).

Thiroredoxin control was not detected by E.canis antiserum (not shown).

Figure 3 shows alignment of ECa28-1 protein (SEQ ID NO.

and ECa28SA2 (partial sequence, SEQ ID NO. 7) and ECa28SA1 (SEQ ID NO. E. chaffeensis P28 (SEQ ID NO. E. chaffeensis OMP-1 family (SEQ ID NOs: 10-14) and C. ruminantium MAP-1 (SEQ ID NO.

amino acid sequences. The ECa28-1 amino acid sequence is presented as the consensus sequence. Amino acids not shown are identical to ECa28-1 and are represented by a dot. Divergent amino acids are shown with the corresponding one letter abbreviation. Gaps introduced for maximal alignment of the amino acid sequences are denoted with a dash. Variable regions are underlined and denoted (VR1, VR2, VR3, and VR4). The arrows indicate the predicted signal peptidase cleavage site for the signal peptide.

Figure 4 shows phylogenetic relatedness of E. canis ECa28- 1 with the ECa28SA2 (partial sequence) and ECa28SA1, 6 members of the E.chaffeensis omp-1 multiple gene family, and C. rumanintium map-1 from deduced amino acid sequences utilizing unbalanced tree WO 00/32745 PCT/US99/28075 construction. The length of each pair of branches represents the distance between the amino acid sequence of the pairs. The scale measures the distance between sequences.

Figure 5 shows Southern blot analysis of E. canis genomic DNA completely digested with six individual restriction enzymes and hybridized with a ECa28-1 DIG-labeled probe (Lanes DIG-labeled molecular weight markers (Lanes 1 and 8).

Figure 6 shows comparison of predicted protein characteristics of ECa28-1 (Jake strain) and E. chaffeensis P28 (Arkansas strain). Surface probability predicts the surface residues by using a window of hexapeptide. A surface residue is any residue with a >2.0 nm 2 of water accessible surface area. A hexapeptide with a value higher than 1 was considered as surface region. The antigenic index predicts potential antigenic determinants. The regions with a value above zero are potential antigenic determinants. T-cell motif locates the potential T-cell antigenic determinants by using a motif of amino acids with residue 1-glycine or polar, residue 2-hydrophobic, residue 3-hydrophobic, residue 4-hydrophobic or proline, and residue or glycine. The scale indicates amino acid positions.

Figure 7 shows nucleic acid sequences and deduced amino acid sequences of the E. canis 28-kDa protein genes ECa28SA2 (nucleotide 1-849: SEQ ID No. 3; amino acid sequence: SEQ ID No. 4) and ECa28SA3 (nucleotide 1195-2031: SEQ ID No. 5; amino acid sequence: SEQ ID No. 6) including intergenic noncoding sequences (NC2, nucleotide 850-1194: SEQ ID No. 31). The ATG start codon and termination condons are shown in bold.

Figure 8 shows schematic of the five E. canis 28-kDa protein gene locus (5.592-Kb) indicating genomic orientation and intergenic noncoding regions (28NC1-4). The 28-kDa protein genes shown in Locus 1 and 2 (shaded) have been described (McBride et al., WO 00/32745 PCT/US99/28075 1999; Reddy et al.. 1998; Ohashi et al.. 1998). The complete sequence of ECaSA2 and a new 28-kDa protein gene designated (ECa28SA3 unshaded) was sequenced. The noncoding intergenic regions (28NC2- 3) between ECaSA2. ECa28SA3 and ECa28-1 were completed joining the previously unlinked loci 1 and 2.

Figure 9 shows phylogenetic relatedness of the five E canis 28-kDa protein gene members based on amino acid sequences utilizing unbalanced tree construction. The length of each pair of branches represents the distance between amino acid pairs. The scale measures the distance beteween sequences.

Figure 10 shows alignment of E. canis 28-kDa protein gene intergenic noncoding nucleic acid sequences (SEQ ID Nos. 33). Nucleic acids not shown, denoted with a dot are identical to noncoding region 1 (28NC1). Divergence is shown with the corresponding one letter abbreviation. Gaps introduced for maximal alignment of the amino acid sequences are denoted with a dash Putative transcriptional promoter regions (-10 and -35) and ribosomal binding site (RBS) are boxed.

DETAILED DESCRIPTION OF THE INVENTION The present invention describes cloning, sequencing and expression of homologous genes encoding a 30-kilodalton (kDa) protein of Ehrlichia canis. A comparative molecular analysis of homologous genes among seven E. canis isolates and the E. chaffeensis omp-1 multigene family was also performed. Two new 28-kDa protein genes are identified, ECa28-1 and ECa28SA3. ECa28-1 has an 834-bp open reading frame encoding a protein of 278 amino acids (SEQ ID No. 2) with a predicted molecular mass of 30.5-kDa. An N-terminal signal sequence was identified suggesting that the protein is post- WO 00/32745 PCTIUS99/28075 translationally modified to a mature protein of 27.7-kDa. ECa28SA3 has an 840-bp open reading frame encoding a 280 amino acid protein (SEQ ID No. 6).

Using PCR to amplify 28-kDa protein genes of E. canis, a previously unsequenced region of Eca28SA2 was completed. Sequence analysis of ECa28SA2 revealed an 849-bp open reading frame encoding a 283 amino acid protein (SEQ ID No. PCR amplification using primers specific for 28-kDa protein gene intergenic noncoding regions linked two previously separate loci, identifying a single locus (5.592kb) containing all five 28-kDa protein genes. The five 28-kDa proteins were predicted to have signal peptides resulting in mature proteins, and had amino acid homology ranging from 51 to 72%.

Analysis of intergenic regions revealed hypothetical promoter regions for each gene, suggesting that these genes may be independently and differentially expressed. Intergenic noncoding regions (28NC1-4) ranged in size from 299 to 355-bp, and were 48 to 71% homologous.

The present invention is directed to two new homologous 28-kDa protein genes in Ehrlichia canis, Eca28-1 and ECa28SA3, and a complete sequence of previously partially sequenced ECa28SA2. Also disclosed is a multigene locus encoding all five homologous 28-kDa outer membrane proteins of Ehrlichia canis.

In one embodiment of the present invention, there are provided DNA sequences encoding a 30-kDa immunoreactive protein of Ehrlichia canis. Preferably, the protein has an amino acid sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4 and SEQ ID No. 6. and the gene has a nucleic acid sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3 and SEQ ID No. and is a member of a polymorphic multiple gene family. More preferably, the protein has an N-terminal signal sequence which is cleaved after post-translational process resulting in the production of WO 00/32745 PCT[US99/28075 a mature 28-kDa protein. Still preferably, the DNAs encoding 28-kDa proteins are contained in a single multigene locus, which has the size of 5.592 kb and encodes all five homologous 28-kDa proteins of Ehrlichia canis.

In another embodiment of the present invention, there is provided an expression vector comprising a gene encoding a 28-kDa immunoreactive protein of Ehrlichia canis and capable of expressing the gene when the vector is introduced into a cell.

In still another embodiment of the present invention, there is provided a recombinant protein comprising an amino acid sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4 and SEQ ID No. 6. Preferably, the amino acid sequence is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3 and SEQ ID No. 5. Preferably, the recombinant protein comprises four variable regions which are surface exposed, hydrophilic and antigenic. Still preferably, the recombinant protein is an antigen.

In yet another embodiment of the present invention, there is provided a method of producing the recombinant protein, comprising the steps of obtaining a vector that comprises an expression region comprising a sequence encoding the amino acid sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4 and SEQ ID No. 6 operatively linked to a promoter; transfecting the vector into a cell; and culturing the cell under conditions effective for expression of the expression region.

The invention may also be described in certain embodiments as a method of inhibiting Ehrlichia canis infection in a subject comprising the steps of: identifying a subject suspected of being exposed to or infected with Ehrlichia canis; and administering a composition comprising a 28-kDa antigen of Ehrlichia canis in an WO 00/32745 PCT/US99/28075 amount effective to inhibit an Ehrlichia canis infection. The inhibition may occur through any means such as, i.e. the stimulation of the subject's humoral or cellular immune responses, or by other means such as inhibiting the normal function of the 28-kDa antigen, or even competing with the antigen for interaction with some agent in the subject's body.

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, Maniatis, Fritsch Sambrook, "Molecular Cloning: A Laboratory Manual (1982); "DNA Cloning: A Practical Approach," Volumes I and II Glover ed.

1985); "Oligonucleotide Synthesis" Gait ed. 1984); "Nucleic Acid Hybridization" Hames S.J. Higgins eds. (1985)]; "Transcription and Translation" Hames S.J. Higgins eds. (1984)]; "Animal Cell Culture" Freshney, ed. (1986)]; "Immobilized Cells And Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning" (1984).

Therefore, if appearing herein, the following terms shall have the definitions set out below.

A "replicon" is any genetic element plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; capable of replication under its own control.

A "vector" is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.

A "DNA molecule" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, WO 00/32745 PCTIUS99/28075 and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA the strand having a sequence homologous to the mRNA).

A DNA "coding sequence" is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.

Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.

A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a WO 00/32745 PCT/US99/28075 transcription initiation site, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters often, but not always, contain "TATA" boxes and "CAT" boxes. Prokaryotic promoters contain Shine- Dalgarno sequences in addition to the -10 and -35 consensus sequences.

An "expression control sequence" is a INA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.

A "signal sequence" can be included near the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.

The term "oligonucleotide", as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.

The term "primer" as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, in the WO 00/32745 PCT/US99/28075 presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and use the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.

The primers herein are selected to be "substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence or hybridize therewith and thereby form the template for the synthesis of the extension product.

A cell has been "transformed" by exogenous or heterologous DNA when such DNA has been introduced inside the cell.

The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNAhas become integrated into a chromosome so that WO 00/32745 PCTIUS99/28075 it is inherited by daughter cells through chromosome replication.

This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.

Two DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%, and most preferably at least about 90% or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, Maniatis et al., supra; DNA Cloning, Vols. I II, supra; Nucleic Acid Hybridization, supra.

A "heterologous' region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. In another example, coding sequence is a construct where the coding sequence itself is not found in nature a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturallyoccurring mutational events do not give rise to a heterologous region of DNA as defined herein.

WO 00/32745 PCT/US99/28075 The labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to untraviolet light, and others. A number of fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow. A particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.

Proteins can also be labeled with a radioactive element or with an enzyme. The radioactive label can be detected by any of the currently available counting procedures. The preferred isotope may be selected from 3 H, 14C, 32p, 35S, 36C1, 5 1 Cr, 5 7 CO, 5 8 CO, 5 9 Fe, 90y, 125i, 1311, and 1 8 6 Re.

Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques.

The enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, P-glucuronidase, p-D-glucosidase, P-D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase.

U.S. Patent Nos. 3,654,090, 3,850,752, and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.

As used herein, the term "host" is meant to include not only prokaryotes but also eukaryotes such as yeast, plant and animal cells. A recombinant DNA molecule or gene which encodes a 28-kDa immunoreactive protein of Ehrlichia canis of the present invention can WO 00/32745 PCT[US99/28075 be used to transform a host using any of the techniques commonly known to those of ordinary skill in the art. Especially preferred is the use of a vector containing coding sequences for a gene encoding a 28kDa immunoreactive protein of Ehrlichia canis of the present invention for purposes of prokaryote transformation.

Prokaryotic hosts may include E. coli, S. tymphimurium, Serratia marcescens and Bacillus subtilis. Eukaryotic hosts include yeasts such as Pichia pastoris, mammalian cells and insect cells.

In general, expression vectors containing promoter sequences which facilitate the efficient transcription of the inserted DNA fragment are used in connection with the host. The expression vector typically contains an origin of replication, promoter(s), terminator(s), as well as specific genes which are capable of providing phenotypic selection in transformed cells. The transformed hosts can be fermented and cultured according to means known in the art to achieve optimal cell growth.

The invention includes a substantially pure DNA encoding a 28-kDa immunoreactive protein of Ehrlichia canis, a strand of which DNA will hybridize at high stringency to a probe containing a sequence of at least 15 consecutive nucleotides of SEQ ID No. 1 or SEQ ID No. 3 or SEQ ID No. 5. The protein encoded by the DNA of this invention may share at least 80% sequence identity (preferably more preferably 90%, and most preferably 95%) with the amino acids listed in SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6. More preferably, the DNA includes the coding sequence of the nucleotides of SEQ ID No. 1 or SEQ ID No. 3 or SEQ ID No. 5, or a degenerate variant of such a sequence.

The probe to which the DNA of the invention hybridizes preferably consists of a sequence of at least 20 consecutive nucleotides, more preferably 40 nucleotides, even more preferably WO 00/32745 PCT/US99/28075 nucleotides, and most preferably 100 nucleotides or more (up to 100%) of the coding sequence of the nucleotides listed in SEQ ID No. 1 or SEQ ID No. 3 or SEQ ID No. 5 or the complement thereof. Such a probe is useful for detecting expression of the 28-kDa immunoreactive protein of Ehrlichia canis in a human cell by a method including the steps of contacting mRNA obtained from the cell with the labeled hybridization probe; and detecting hybridization of the probe with the mRNA.

This invention also includes a substantially pure DNA containing a sequence of at least 15 consecutive nucleotides (preferably 20, more preferably 30, even more preferably 50, and most preferably all) of the region from the nucleotides listed in SEQ ID No 1 or SEQ ID No. 3 or SEQ ID No. By "high stringency" is meant DNA hybridization and wash conditions characterized by high temperature and low salt concentration, wash conditions of 65°C at a salt concentration of approximately 0.1 x SSC, or the functional equivalent thereof. For example, high stringency conditions may include hybridization at about 42°C in the presence of about 50% formamide; a first wash at about 65°C with about 2 x SSC containing 1% SDS; followed by a second wash at about 65°C with about 0.1 x SSC.

By "substantially pure DNA" is meant DNA that is not part of a milieu in which the DNA naturally occurs, by virtue of separation (partial or total purification) of some or all of the molecules of that milieu, or by virtue of alteration of sequences that flank the claimed DNA. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule a cDNA or a genomic or cDNA fragment produced by polymerase chain reaction WO 00/32745 PCT/US99/28075 (PCR) or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence, a fusion protein. Also included is a recombinant DNA which includes a portion of the nucleotides listed in SEQ ID No. 1 or SEQ ID No. 3 or SEQ ID No.

which encodes an alternative splice variant of a gene encoding a 28kDa immunoreactive protein of Ehrlichia canis.

The DNA may have at least about 70% sequence identity to the coding sequence of the nucleotides listed in SEQ ID No. 1 or SEQ ID No. 3 or SEQ ID No. 5, preferably at least 75% at least and most preferably at least 90%. The identity between two sequences is a direct function of the number of matching or identical positions.

When a subunit position in both of the two sequences is occupied by the same monomeric subunit, if a given position is occupied by an adenine in each of two DNA molecules, then they are identical at that position. For example, if 7 positions in a sequence nucleotides in length are identical to the corresponding positions in a second 10-nucleotide sequence, then the two sequences have sequence identity. The length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 100 nucleotides. Sequence identity is typically measured using sequence analysis software Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705).

The present invention comprises a vector comprising a DNA sequence coding for a which encodes a gene encoding a 28-kDa immunoreactive protein of Ehrlichia canis and said vector is capable of replication in a host which comprises, in operable linkage: a) an origin of replication; b) a promoter; and c) a DNA sequence coding WO 00/32745 PCT/US99/28075 for said protein. Preferably, the vector of the present invention contains a portion of the DNA sequence shown in SEQ ID No. 1 or SEQ ID No. 3 or SEQ ID No. A "vector" may be defined as a replicable nucleic acid construct, a plasmid or viral nucleic acid. Vectors may be used to amplify and/or express nucleic acid encoding a 28-kDa immunoreactive protein of Ehrlichia canis. An expression vector is a replicable construct in which a nucleic acid sequence encoding a polypeptide is operably linked to suitable control sequences capable of effecting expression of the polypeptide in a cell. The need for such control sequences will vary depending upon the cell selected and the transformation method chosen. Generally, control sequences include a transcriptional promoter and/or enhancer, suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation. Methods which are well known to those skilled in the art can be used to construct expression vectors containing appropriate transcriptional and translational control signals. See for example, the techniques described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual (2nd Cold Spring Harbor Press, N.Y. A gene and its transcription control sequences are defined as being "operably linked" if the transcription control sequences effectively control the transcription of the gene. Vectors of the invention include, but are not limited to, plasmid vectors and viral vectors. Preferred viral vectors of the invention are those derived from retroviruses, adenovirus, adeno-associated virus, SV40 virus, or herpes viruses.

By a "substantially pure protein" is meant a protein which has been separated from at least some of those components which naturally accompany it. Typically, the protein is substantially pure when it is at least 60%, by weight, free from the proteins and other WO 00/32745 PCT/US99/28075 naturally-occurring organic molecules with which it is naturally associated in vivo. Preferably, the purity of the preparation is at least more preferably at least 90%, and most preferably at least 99%, by weight. A substantially pure 28-kDa immunoreactive protein of Ehrlichia canis may be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid encoding a 28-kDa immunoreactive protein of Ehrlichia canis; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, column chromatography such as immunoaffinity chromatography using an antibody specific for a 28-kDa immunoreactive protein of Ehrlichia canis, polyacrylamide gel electrophoresis, or HPLC analysis. A protein is substantially free of naturally associated components when it is separated from at least some of those contaminants which accompany it in its natural state.

Thus, a protein which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be, by definition, substantially free from its naturally associated components. Accordingly, substantially pure proteins include eukaryotic proteins synthesized in E. coli, other prokaryotes, or any other organism in which they do not naturally occur.

In addition to substantially full-length proteins, the invention also includes fragments antigenic fragments) of the 28-kDa immunoreactive protein of Ehrlichia canis (SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. As used herein, "fragment," as applied to a polypeptide, will ordinarily be at least 10 residues, more typically at least 20 residues, and preferably at least 30 50) residues in length, but less than the entire, intact sequence. Fragments of the 28kDa immunoreactive protein of Ehrlichia canis can be generated by methods known to those skilled in the art, by enzymatic digestion of naturally occurring or recombinant 28-kDa immunoreactive WO 00/32745 PCT/US99/28075 protein of Ehrlichia canis, by recombinant DNA techniques using an expression vector that encodes a defined fragment of 28-kDa immunoreactive protein of Ehrlichia canis, or by chemical synthesis.

The ability of a candidate fragment to exhibit a characteristic of 28kDa immunoreactive protein of Ehrlichia canis binding to an antibody specific for 28-kDa immunoreactive protein of Ehrlichia canis) can be assessed by methods described herein. Purified 28-kDa immunoreactive protein of Ehrlichia canis or antigenic fragments of 28-kDa immunoreactive protein of Ehrlichia canis can be used to generate new antibodies or to test existing antibodies as positive controls in a diagnostic assay) by employing standard protocols known to those skilled in the art. Included in this invention are polyclonal antisera generated by using 28-kDa immunoreactive protein of Ehrlichia canis or a fragment of 28-kDa immunoreactive protein of Ehrlichia canis as the immunogen in, rabbits. Standard protocols for monoclonal and polyclonal antibody production known to those skilled in this art are employed. The monoclonal antibodies generated by this procedure can be screened for the ability to identify recombinant Ehrlichia canis cDNA clones, and to distinguish them from known cDNA clones.

Further included in this invention are fragments of the 28kDa immunoreactive protein of Ehrlichia canis which are encoded at least in part by portions of SEQ ID No. 1 or SEQ ID No. 3 or SEQ ID No.

products of alternative mRNA splicing or alternative protein processing events, or in which a section of the sequence has been deleted. The fragment, or the intact 28-kDa immunoreactive protein of Ehrlichia canis, may be covalently linked to another polypeptide, e.g. which acts as a label, a ligand or a means to increase antigenicity.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic WO 00/32745 PCT/US99/28075 or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified.

A protein 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 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.

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.

These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 mL of isotonic NaCl solution and either added to 1000mL of WO 00/32745 PCTIUS99/28075 hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

As is well known in the art, a given polypeptide may vary in its immunogenicity. It is often necessary therefore to couple the immunogen a polypeptide of the present invention) with a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and human serum albumin. Other carriers may include a variety of lymphokines and adjuvants such as IL2, IL4, IL8 and others.

Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, mmaleimidobenzoyl-N-hydroxysuccinimide ester, carbo-diimide and bis-biazotized benzidine. It is also understood that the peptide may be conjugated to a protein by genetic engineering techniques that are well known in the art.

As is also well known in the art, immunogenicity to a particular immunogen can be enhanced by the use of non-specific stimulators of the immune response known as adjuvants. Exemplary and preferred adjuvants include complete BCG, Detox, (RIBI, Immunochem Research Inc.) ISCOMS and aluminum hydroxide adjuvant (Superphos, Biosector).

As used herein the term "complement" is used to define the strand of nucleic acid which will hybridize to the first nucleic acid sequence to form a double stranded molecule under stringent conditions. Stringent conditions are those that allow hybridization between two nucleic acid sequences with a high degree of homology, WO 00/32745 PCT/US99/28075 but precludes hybridization of random sequences. For example, hybridization at low temperature and/or high ionic strength is termed low stringency and hybridization at high temperature and/or low ionic strength is termed high stringency. The temperature and ionic strength of a desired stringency are understood to be applicable to particular probe lengths, to the length and base content of the sequences and to the presence of formamide in the hybridization mixture.

As used herein, the term "engineered" or "recombinant" cell is intended to refer to a cell into which a recombinant gene, such as a gene encoding an Ehrlichia chaffeensis antigen has been introduced. Therefore, engineered cells are distinguishable from naturally occurring cells which do not contain a recombinantly introduced gene. Engineered cells are thus cells having a gene or genes introduced through the hand of man. Recombinantly introduced genes will either be in the form of a cDNA gene, a copy of a genomic gene, or will include genes positioned adjacent to a promoter not naturally associated with the particular introduced gene. In addition, the recombinant gene may be integrated into the host genome, or it may be contained in a vector, or in a bacterial genome transfected into the host cell.

The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.

EXAMPLE 1 Ehrlichiae and Purification Ehrlichia canis (Florida strain and isolates Demon, DJ, Jake, and Fuzzy) were provided by Dr. Edward Breitschwerdt, (College WO 00/32745 PCT[US99/28075 of Veterinary Medicine, North Carolina State University, Raleigh, NC).

E. canis (Louisiana strain) was provided by Dr. Richard E. Corstvet (School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA) and E. canis (Oklahoma strain) was provided by Dr.

Jacqueline Dawson (Centers for Disease Control and Prevention, Atlanta, GA). Propagation of ehrlichiae was performed in DH82 cells with DMEM supplemented with 10% bovine calf serum and 2 mM L, glutamine at 37oC. The intracellular growth in DH82 cells was monitored by presence of E. canis morulae using general cytologic staining methods. Cells were harvested when 100% of the cells were infected with ehrlichiae and were then pelleted in a centrifuge at 17,000 x g for 20 min. Cell pellets were disrupted with a Braun-Sonic 2000 sonicator twice at 40W for 30 sec on ice. Ehrlichiae were purified as described previously (Weiss et al., 1975). The lysate was loaded onto discontinuous gradients of 42%-36%-30% renografin, and centrifuged at 80,000 x g for 1 hr. Heavy and light bands containing ehrlichiae were collected and washed with sucrose-phosphateglutamate buffer (SPG, 218 mM sucrose, 3.8 mM KH2PO4, 7.2 mM

K

2

HPO

4 4.9 mM glutamate, pH 7.0) and pelleted by centrifugation.

EXAMPLE 2 Nucleic Acid Preparation Ehrlichia canis genomic DNA was prepared by resuspending the renografin-purified ehrlichiae in 600 g1 of 10 mM Tris-HCl buffer (pH 7.5) with 1% sodium dodecyl sulfate (SDS, w/v) and 100 ng/ml of proteinase K as described previously (McBride et al., 1996). This mixture was incubated for 1 hr at 560 C, and the nucleic acids were extracted twice with a mixture of phenol/chloroform/isoamyl alcohol (24:24:1). DNA was pelleted by WO 00/32745 PCT/US99/28075 absolute ethanol precipitation, washed once with 70% ethanol, dried and resuspended in 10mM Tris (pH Plasmid DNA was purified by using High Pure Plasmid Isolation Kit (Boehringer Mannheim, Indianapolis, IN), and PCR products were purified using a QIAquick PCR Purification Kit (Qiagen, Santa Clarita, CA).

EXAMPLE3 PCR Amplification of the F. canis 28-kDa protein Genes Regions of the E. canis ECa28-1 gene selected for PCR amplification were chosen based on homology observed in the consensus sequence generated from Jotun-Hein aligorithm alignment of E. chaffeensis p28 and Cowdria ruminantium map-1 genes. Forward primer 793 (5-GCAGGAGCTGTTGGTTACTC-3') (SEQ ID NO. 16) and reverse primer 1330 (5'-CCTTCCTCCAAGTTCTATGCC-3') (SEQ ID NO.

17) corresponded to nucleotides 313-332 and 823-843 of C.

ruminantium MAP-1 and 307-326 and 834-814 of E. chaffeensis P28.

E. canis (a North Carolina isolate, Jake) DNA was amplified with primers 793 and 1330 with a thermal cycling profile of 95°C for 2 min, and 30 cycles of 95°C for 30 sec, 620 C for 1 min, 72 0 C for 2 min followed by a 72 0 C extension for 10 min and 4 0 C hold. PCR products were analyzed on 1% agarose gels. This amplified PCR product was sequenced directly with primers 793 and 1330.

Primers specific for ECa28SA2 gene designated 46f ATATACTTCCTACCTAATGTCTCA-3', SEQ ID No. 18) and primer 1330 (SEQ ID No. 17) were used to amplify the targeted region. The amplified product was gel purified and cloned into a TA cloning vector (Invitrogen, Santa Clarita, CA). The clone was sequenced bidirectionally with primers: M13 reverse from the vector, 46f, ECa28SA2 (5'-AGTGCAGAGTCTTCGGTTTC-3', SEQ ID No. 19), ECa5.3 WO 00/32745 PCT/US99/28075 (5'-GTTACTTGCGGAGGACAT-3', SEQ ID No. 20). DNA was amplified with a thermal cycling profile of 95°C for 2 min, and 30 cycles of for 30 sec, 48 0 C for 1 min, 72 0 C for 1 min followed by a 72 0

C

extension for 10 min and 4 0 C hold.

EXAMPL,E4 Sequencing Unknown 5' and 3' Regions of the RCa28-1 Gene The full length sequence of ECa28-1 was determined using a Universal GenomeWalker Kit (CLONTECH, Palo Alto, CA) according to the protocol supplied by the manufacturer. Genomic E. canis (Jake isolate) DNA was digested completely with five restriction enzymes (DraI, EcoRV, PvuII, ScaI, StuI) which produce blunt-ended DNA. An adapter (API) supplied in the kit was ligated to each end of E. canis DNA. The genomic libraries were used as templates to find the unknown DNA sequence of the ECa28-1 gene by PCR using a primer complementary to a known portion of the ECa28-1 sequence and a primer specific for the adapter API. Primers specific for ECa28-1 used for genome walking were designed from the known DNA sequence derived from PCR amplification of ECa28-1 with primers 793 (SEQ ID NO. 16) and 1330 (SEQ ID NO. 17). Primers 394 GCATTTCCACAGGATCATAGGTAA-3'; nucleotides 687-710, SEQ ID NO.

21) and 394C (5'-TTACCTATGATCCTGT GGAAATGC-3; nucleotides 710-687, SEQ ID NO. 22) were used in conjunction with supplied primer API to amplify the unknown 5' and 3' regions of the ECa28-1 gene by PCR. A PCR product corresponding to the 5' region of the ECa28-1 gene amplified with primers 394C and API (2000-bp) was sequenced unidirectionally with primer 793C ACCAACAGCTCCTGC-3', SEQ ID No. 23). A PCR product corresponding WO 00/32745 PCT/US99/28075 to the 3' region of the ECa28-1 gene amplified with primers 394 and AP1 (580-bp) was sequenced bidirectionally with the same primers.

Noncoding regions on the 5' and 3' regions adjacent to the open reading frame were sequenced, and primers EC28OM-F TCTACTTTGCACTTCC ACTATTGT-3', SEQ ID NO. 24) and EC280M-R ATTCTTTTGCCACTATTT TTCTTT-3', SEQ ID NO. 25) complementary to these regions were designed in order to amplify the entire ECa28-1 gene.

EXAMPLE Sequencing of E. canis isolates DNA was sequenced with an ABI Prism 377 DNA Sequencer (Perkin- Elmer Applied Biosystems, Foster City, CA). The entire Eca28- 1 genes of seven E. canis isolates (four from North Carolina, and one each from Oklahoma, Florida, and Louisiana) were amplified by PCR with primers EC280M-F (SEQ ID No. 24) and EC280M-R (SEQ ID No.

with a thermal cycling profile of 95°C for 5 minutes, and 30 cycles of 95°C for 30 seconds, 62"C for 1 minutes, and 720C for 2 minutes and a 72oC extension for 10 minutes. The resulting PCRproducts were bidirectionally sequenced with the same primers.

EXAMPLE 6 Cloning and Expression of E canis ECa28-1 The entire E. canis ECa28-1 gene was PCR-amplified with primers-EC280M-F and EC28OM-R and cloned into pCR2.1-TOPO TA cloning vector to obtain the desired set of restriction enzyme cleavage sites (Invitrogen, Carlsbad, CA). The insert was excised from pCR2.1- WO 00/32745 PCT/US99/28075 TOPO with BstX 1 and ligated into pcDNA 3.1 eukaryotic expression vector (Invitrogen, Carlsbad, CA) designated pcDNA3.1/EC28 for subsequent studies. The pcDNA3.1/EC28 plasmid was amplified, and the gene was excised with a KpnI-Xbal double digestion and directionally ligated into pThioHis prokaryotic expression vector (Invitrogen, Carlsbad, CA). The clone (designated pThioHis/EC28) produced a recombinant thioredoxin fusion protein in Escherichia coli BL21. The recombinant fusion protein was crudely purified in the insoluble phase by centrifugation. The control thioredoxin fusion protein was purified from soluble cell lysates under native conditions using nickel-NTA spin columns (Qiagen, Santa Clarita, CA).

EXAMPLE 7 Western Immunoblot Analysis Recombinant E. canis ECa28-1 fusion protein was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 4-15% Tris- HC1 gradient gels (Bio-Rad, Hercules, CA) and transferred to pure nitrocellulose (Schleicher Schuell, Keene, NH) using a semi-dry transfer cell (Bio-Rad, Hercules, CA). The membrane was incubated with convalescent phase antisera from an E. canis-infected dog diluted 1:5000 for 1 hour, washed, and then incubated with an anti-canine IgG (H L) alkaline phosphatase-conjugated affinity-purified secondary antibody at 1:1000 for 1 hour (Kirkegaard Perry Laboratories, Gaithersburg, MD). Bound antibody was visualized with 5-bromo-4chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) substrate (Kirkegaard Perry Laboratories, Gaithersburg, MD).

WO 00/32745 PCT/US99/28075

EXAMPLE

Southern Blot Analysis To determine if multiple genes homologous to the ECa28-1 gene were present in the E. canis genome, a genomic Southern blot analysis was performed using a standard procedure (Sambrook et al.

1989). E. canis genomic DNA digested completely with each of the restriction enzymes BanII, EcoRV, HaeII, KpnI and Spel, which do not cut within the ECa28-1 gene, and AseI which digests ECa28-1 at nucleotides 34, 43 and 656. The probe was produced by PCR amplification with primers EC280M-F and EC280M-R and digoxigenin (DIG)-labeled deoxynucleotide triphosphates (dNTPs) (Boehringer Mannheim, Indianapolis, IN) and digested with AseI. The digested probe (566-bp) was separated by agarose gel electrophoresis, gelpurified and then used for hybridization. The completely digested genomic E. canis DNA was electrophoresed and transferred to a nylon membrane (Boehringer Mannheim, Indianapolis, IN) and hybridized at 0 C for 16 hr with the ECa28-1 gene DIG-labeled probe in DIG Easy Hyb buffer according to the manufacturer's protocol (Boehringer Mannheim, Indianapolis, IN). Bound probe was detected with a anti- DIG alkaline phosphatase-conjugated antibody and a luminescent substrate (Boehringer Mannheim, Indianapolis, IN) and exposed to BioMax scientific imaging film (Eastman Kodak, Rochester, NY).

EXAMPLE 9 Sequence Analysis and Comparasion E. chaffeensis p28 and C. ruminantium map-] DNA sequences were obtained from the National Center of Biotechnology Information (NCBI) (World Wide Web site at URL: WO 00/32745 PCT/US99/28075 http://www.ncbi.nlm.nih.gov/Entrez). Nucleotide and deduced amino acid sequences, and protein and phylogenetic analyses were performed with LASERGENE software (DNASTAR, Inc., Madison, WI).

Analysis of post-translational processing was performed by the method of McGeoch and von Heijne for signal sequence recognition using the PSORTprogram (McGeoch, 1985; von Heijne, 1986) (World Wide Web site at URL: PRIVATE HREF= "http://www.imcb.osakau.ac.jp/nakai/form.htm", MACROBUTTON HtmlResAnchor http://www.imcb.osaka-u.ac.jp/nakai/form.htm).

GenBank accession numbers for nucleic acid and amino acid sequences of the E. canis ECa28-1 genes described in this study are: Jake, AF082744; Louisiana, AF082745; Oklahoma, AF082746; Demon, AF082747; DJ, AF082748; Fuzzy, AF082749; Florida, AF082750.

Sequence analysis of ECa28-1 from seven different strains of E. canis was performed with primers designed to amplify the entire gene. Analysis revealed the sequence of this gene was conserved among the isolates from North Carolina (four), Louisiana, Florida and Oklahoma.

EXAMPLE PCR Amplification. Cloning. Sequencing and Expression of ECa28-1 Alignment of nucleic acid sequences from E. chaffeensis p 2 8 and Cowdria ruminantium map-] using the Jotun-Hein aligorithm produced a consensus sequence with regions of high homology These homologous regions (nucleotides 313-332 and 823- 843 of C. ruminantium map-i; 307-326 and 814-834 of E. chaffeensis p28) were targeted as primer annealing sites for PCR amplification.

PCR amplification of the E. canis ECa28-1 and E. chaffeensis p28 gene WO 00/32745 PCT/US99/28075 was accomplished with primers 793 and 1330, resulting in a 518-bp PCR product. The nucleic acid sequence of the E. canis PCR product was obtained by sequencing the product directly with primers 793 and 1330. Analysis of the sequence revealed an open reading frame encoding a protein of 170 amino acids, and alignment of the 518-bp sequence obtained from PCR amplification of E. canis with the DNA sequence of E. chaffeensis p28 gene revealed a similarity greater than indicating that the genes were homologous. Adapter PCR with primers 394 and 793C was performed to determine the 5' and 3' segments of the sequence of the entire gene. Primer 394 produced four PCR products (3-kb, 2-kb, 1-kb, and 0.8-kb), and the 0.8-bp product was sequenced bidirectionally using primers 394 and API.

The deduced sequence overlapped with the 3' end of the 518-bp product, extending the open reading frame 12-bp to a termination codon. An additional 625-bp of non-coding sequence at the 3' end of the ECa28-1 gene was also sequenced. Primer 394C was used to amplify the 5' end of the ECa28-1 gene with supplied primer API.

Amplification with these primers resulted in three PCR products (3.3, 3-kb, and 2-kb). The 2-kb fragment was sequenced unidirectionally with primer 793C. The sequence provided the putative start codon of the ECa28-1 gene and completed the 834-bp open reading frame encoding a protein of 278 amino acids. An additional 144-bp of readable sequence in the 5' noncoding region of the ECa28-1 gene was generated. Primers EC280M-F and EC28OM-R were designed from complementary non-coding regions adjacent to the ECa28-1 gene.

The PCR product amplified with these primers was sequenced directly with the same primers. The complete DNA sequence (SEQ ID NO. 1) for the E. canis ECa28-1 gene is shown in Figure 1. The ECa28-1 PCR fragment amplified with these primers contained the entire open reading frame and 17 additional amino WO 00/32745 PCT/US99/28075 acids from the 5' non-coding primer region. The gene was directionally subcloned into pThioHis expression vector, and E. coli (BL21) were transformed with this construct. The expressed ECa28-1thioredoxin fusion protein was insoluble. The expressed protein had an additional 114 amino acids associated with the thioredoxin, amino acids for the enterokinase recognition site, and 32 amino acids from the multiple cloning site and 5' non-coding primer region at the N-terminus. Convalescent-phase antiserum from an E. canis infected dog recognized the expressed recombinant fusion protein, but did not react with the thioredoxin control (Figure 2).

EXAMPLE 11 Sequence Homology The nucleic acid sequence of ECa28-1 (834-bp) and the E chaffeensis omp-] family of genes including signal sequences (ECa28- 1, omp-1A, B, C, D, E, and F) were aligned using the Clustal method to examine homology between these genes (alignment not shown).

Nucleic acid homology was equally conserved between ECa28- 1, and E. chaffeensis p28 and omp-lF. Other putative outer membrane protein genes in the E. chaffeensis omp-1 family, omp-1 D omp-lE omp-1C Cowdria ruminantium map-1 E. canis 28-kDa protein 1 gene and 28-kDa protein 2 gene (partial) were also homologous to ECa28-1. E chaffeensis omp-lB had the least nucleic acid homology with E.Ca28-1.

Alignment of the predicted amino acid sequences of ECa28-1 (SEQ ID NO. 2) and E. chaffeensis P28 revealed amino acid substitutions resulting in four variable regions Substitutions or deletions in the amino acid sequence and the locations of variable WO 00/32745 PCTIUS99/28075 regions of ECa28-1 and the E. chaffeensis OMP-1 family were identified (Figure Amino acid comparison including the signal peptide revealed that ECa28-1 shared the most homology with OMP-1F (68%) of the E. chaffeensis OMP-1 family, followed by E. chaffeensis P28 OMP-1E OMP-1D OMP-IC Cowdria ruminantium MAP-1 E. canis 28-kDa protein 1 and 28-kDa protein 2 (partial) and OMP-1B The phylogenetic relationships based on amino acid sequences show that ECa28-1 and C. ruminantium MAP-1, E chaffeensis OMP-1 proteins, and E. canis 28-kDa proteins 1 and 2 (partial) are related (Figure 4).

EXAMPLE 12 Predicted Surface Probability and Immunoreactivity Analysis of E. canis ECa28-1 using hydropathy and hydrophilicity profiles predicted surface-exposed regions on ECa28-1 (Figure Eight major surface-exposed regions consisting of 3 to 9 amino acids were identified on ECa28-1 and were similar to the profile of surface-exposed regions on E. chaffeensis P28 (Figure Five of the larger surface-exposed regions on ECa28-1 were located in the Nterminal region of the protein. Surface-exposed hydrophilic regions were found in all four of the variable regions of ECa28-1. Ten T-cell motifs were predicted in the ECa28-1 using the Rothbard-Taylor aligorithm (Rothbard and Taylor, 1988), and high antigenicity of the ECa28-1 was predicted by the Jameson-Wolf antigenicity aligorithm (Figure 6) (Jameson and Wolf, 1988). Similarities in antigenicity and T-cell motifs were observed between ECa28-1 and E. chaffeensis P28.

WO 00/32745 PCT/US99/28075 EXAMPLE 13 Detection of Homologons Genomic Copies of ECa28-1 Gene Genomic Southern blot analysis of E. canis DNA completely digested independently with restriction enzymes BanII, EcoRV, HaeII, KpnI, SpeI, which do not have restriction endonuclease sites in the ECa28-1 gene, and Asel, which has internal restriction endonuclease sites at nucleotides 34, 43 and 656, revealed the presence of at least three homologous ECa28-1 gene copies (Figure Although ECa28-1 has internal Ase I internal restriction sites, the DIG-labeled probe used in the hybridization experiment targeted a region of the gene within a single DNA fragment generated by the Asel digestion of the gene.

Digestion with Asel produced 3 bands (approximately 566-bp, 850 bp, and 3-kb) that hybridized with the ECa28-1 DNA probe indicating the presence of multiple genes homologous to ECa28-1 in the genome.

Digestion with EcoRV and SpeI produced two bands that hybridized with the ECa28-1 gene probe.

EXAMPLE 14 Identification of 28-kDa Protein Gene L.ocus Specific primers designated ECaSA3-2 GGTTATAGTATAAGTT-3', SEQ ID No. 26) corresponding to regions within ECa28SA3 and primer 793C (SEQ ID No. 23) which anneals to a region with ECa28-1 were used to amplify the intergenic region between gene SA3 and ECa28-1. The 800-bp product was sequenced with the same primers. DNA was amplified with a thermal cycling profile of 95°C for 2 min, and 30 cycles of 95°C for 30 sec, 50 0 C for 1 min, 72°C for 1 min followed by a 72°C extension for 10 min and 4°C hold.

WO 00/32745 PCT/US99/28075 EXAMPLE PCR Amplification of 28-kDa Protein Genes and Identification of the Multiple Gene Locus In order to specifically amplify possible unknown genes downstream of ECa28SA2, primer 46f specific for ECa28SA2, and primer 1330 which targets a conserved region on the 3' end of ECa28- 1 gene were used for amplification. A 2-kb PCR product was amplified with these primers that contained 2 open reading frames. The first open reading frame contained the known region of gene, ECaSA2, and a previously unsequenced 3' portion of the gene. Downstream from ECaSA2 an additional non identical, but homologous 28-kDa protein gene was found, and designated ECa28SA3. The two known loci were joined by amplification with primer SA3-2 specific for the 3' end of ECa28SA3 gene was used in conjunction with a reverse primer 793C, which anneals at 5' end of ECa28-1. An 800-bp PCR product was amplified which contained the 3' end of Eca28SA3, the intergenic region between ECa28SA3 and ECa28-1 (28NC3) and the 5' end of Eca28-1, joining the previously separate loci (Figure The 849-bp open reading frame of ECa28SA2 encodes a 283 amino acid protein, and ECa28SA3 has an 840-bp open reading frame encoding a 280 amino acid protein. The intergenic noncoding region between ECa28SA3 and ECa28-1 was 345-bp in length (Figures 7 and 8) EXAMPLE 16 Nucleic and Amino Acid Homology The nucleic and amino acid sequences of all five E. canis 28-kDa protein genes were aligned using the Clustal method to examine the homology between these genes. The nucleic acid WO 00/32745 PCT/US99/28075 homology ranged from 58 to 75% and a similar amino acid homology of ranging from 67 to 72% was observed between the E. canis 28-kDa protein gene members (Figure 9).

EXAMPLE 17 Transcriptional Promoter Regions The intergenic regions between the 28-kDa protein genes were analyzed for promoter sequences by comparison with consensus Escherichia coli promoter regions and a promoter from E. chaffeensis (Yu et al., 1997; McClure, 1985).

Putative promoter sequences including RBS, -10 and regions were identified in 4 intergenic sequences corresponding to genes ECa28SA2, ECa28SA3, ECa28-1, and ECa28-2 (Figure 10). The upstream noncoding region of ECa28SAl is not known and was not analyzed.

EXAMPLE 18 N-Terminal Signal Sequence The amino acid sequence analysis revealed that entire E.canis ECa28-1 has a deduced molecular mass of 30.5-kDa and the entire ECa28SA3 has a deduced molecular mass of 30.7-kDa. Both proteins have a predicted N-terminal signal peptide of 23 amino acids (MNCKKIL1TTALMSLMYYAPSIS, SEQ ID No. 27), which is similar to that predicted for E. chaffeensis P28 (MNYKKILITSALISLISSLPGV SFS, SEQ ID NO. 28), and the OMP-1 protein family (Yu et al., 1998; Ohashi et al., 1998b). A preferred cleavage site for signal peptidases (SIS; Ser-X- Ser) (Oliver, 1985) is found at amino acids 21, 22, and 23 of ECa28-1.

An additional putative cleavage site at amino acid position WO 00/32745 PCT/US99/28075 (MNCKKILITTALISLMYSIPSISSFS, SEQ ID NO. 29) identical to the predicted cleavage site of E. chaffeensis P28 (SFS) was also present, and would result in a mature ECa28-1 with a predicted molecular mass of 27.7-kDa. Signal cleavage site of the previously reported partial sequence of ECa28SA2 is predicted at amino acid 30. However, signal sequence analysis predicted that ECa28SAl had an uncleavable signal sequence.

Summary Proteins of similar molecular mass have been identified and cloned from multiple rickettsial agents including E. canis, E chaffeensis, and C. ruminantium (Reddy et al., 1998; Jongejan et al., 1993; Ohashi et al., 1998). A single locus in Ehrlichia chaffeensis with 6 homologous p28 genes, and 2 loci in E. canis, each containing some homologous 28-kDa protein genes have been previously described.

The present invention demonstrated the cloning, expression and characterization of genes encoding a mature 28-kDa protein of E. canis that are homologous to the omp-1 multiple gene family of E. chaffeensis and the C. ruminantium map-1 gene. Two new 28-kDa protein genes were identidfied, Eca28-1 and ECa28SA3.

Another E.canis 28-kDa protein gene, ECa28SA2, partially sequenced previously (Reddy et al., 1998), was sequenced completely in the present invention. Also disclosed is the identification and characterization of a single locus in E.canis containing all five E.canis 28-kDa protein genes.

The E.canis 28-kDa protein are homologous to E.chaffeensis OMP-1 family and the MAP-I protein of C. rumanintium.

The most homologous E. canis 28-kDa proteins (ECa28SA3, ECa28-1 and ECa28-2) are sequentially arranged in the locus. Homology of these proteins ranged from 67.5% to 72.3%. Divergence among these 28-kDa proteins was 27.3% to 38.6%. E. canis 28-kDa proteins WO 00/32745 PCTIUS99/28075 ECa28SAI and ECa28SA2 were the least homologous with homology ranging from 50.9% to 59.4% and divergence of 53.3 to 69.9%.

Differences between the genes lies primarily in the four hypervariable regions and suggests that these regions are surface exposed and subject to selective pressure by the immune system. Conservation of ECa28-1 among seven E. canis isolates has been reported (McBride et al., 1999), suggesting that E.canis may be clonal in North America.

Conversely, significant diversity of p2 8 among E. chaffeensis isolates has been reported (Yu et al., 1998).

All of the E. canis 28-kDa proteins appear to be post translationally processed from a 30-kD protein to a mature 28-kD protein. Recently, a signal sequence was identified on E. chaffeensis P28 (Yu et al., 1998), and N-terminal amino acid sequencing has verified that the protein is post-translationally processed resulting in cleavage of the signal sequence to produce a mature protein (Ohashi et al., 1998). The leader sequences of OMP-IF and OMP-1E have also been proposed as leader signal peptides (Ohashi et al., 1998). Signal sequences identified on E. chaffeensis OMP-lF, OMP-1E and P28 are homologous to the leader sequence of E. canis 28-kDa protein.

Promoter sequences for the p28 genes have not been determined experimentally, but putative promoter regions were identified by comparison with consensus sequences of the RBS, -10 and promoter regions of E. coli and other ehrlichiae (Yu et al., 1997; McClure, 1985). Such promoter sequences would allow each gene to potentially be transcribed and translated, suggesting that these genes may be differentially expressed in the host. Persistence of infection in dogs may be related to differential expression of p28 genes resulting in antigenic changes in vivo, thus allowing the organism to evade the immune response.

WO 00/32745 PCTUS99/28075 The E. canis 28-kda protein genes were found to exhibit nucleic acid and amino acid sequence homology with the E chaffeensis omp-1 gene family and C. ruminantiunm map-1 gene.

Previous studies have identified a 30-kDa protein of E. canis that reacts with convalescent phase antisera against E. chaffeensis, but was believed to be antigenically distinct (Rikihisa et al., 1994). Findings based on comparison of amino acid substitutions in four variable regions of E. canis 28-kDa proteins support this possibility. Together these findings also suggest that the amino acids responsible for the antigenic differences between E. canis and E. chaffeensis P28 are located in these variable regions and are readily accessible to the immune system. It was reported that immunoreactive peptides were located in the variable regions of the 28-kDa proteins of C.

ruminantium, E. chaffeensis and E. canis (Reddy et al., 1998). Analysis of E. canis and E. chaffeensis P28 revealed that all of the variable regions have predicted surface-exposed amino acids. A study in dogs demonstrated lack of cross protection between E. canis and E chaffeensis (Dawson and Ewing, 1992). This observation may be related to antigenic differences in the variable regions of P28 as well as in other immunologically important antigens of these ehrlichial species. Another study found that convalescent phase human antisera from E. chaffeensis-infected patients recognized 29/28-kDa protein(s) of E. chaffeensis and also reacted with homologous proteins of E. canis (Chen et al., 1997). Homologous and crossreactive epitopes on the E canis 28-kDa protein and E. chaffeensis P28 appear to be recognized by the immune system.

E. canis 28-kDa proteins may be important immunoprotective antigens. Several reports have demonstrated that the 30-kDa antigen of E. canis exhibits strong immunoreactivity (Rikihisa et al., 1994; Rikihisa et al., 1992). Antibodies in WO 00/32745 PCT/US99/28075 convalescent phase antisera from humans and dogs have consistently reacted with proteins in this size range from E. chaffeensis and E canis, suggesting that they may be important immunoprotective antigens (Rikihisa et al., 1994; Chen et al., 1994; Chen et al., 1997). In addition, antibodies to 30, 24 and 21-kDa proteins developed early in the immune response to E. canis (Rikihisa et al., 1994; Rikihisa et al., 1992), suggesting that these proteins may be especially important in the immune responses in the acute stage of disease. Recently, a family of homologous genes encoding outer membrane proteins with molecular masses of 28-kDa have been identified in E. chaffeensis, and mice immunized with recombinant E. chaffeensis P28 appeared to have developed immunity against homologous challenge (Ohashi et al., 1998). The P28 of E. chaffeensis has been demonstrated to be present in the outer membrane, and immunoelectron microscopy has localized the P28 on the surface on the organism, and thus suggesting that it may serve as an adhesin (Ohashi et al., 1998). It is likely that the 28-kDa proteins of E. canis identified in this study have the same location and possibly serve a similar function.

Comparison of ECa28-1 from different strains of E. canis revealed that the gene is apparently completely conserved. Studies involving E. chaffeensis have demonstrated immunologic and molecular evidence of diversity in the ECa28-1. Patients infected with E. chaffeensis have variable immunoreactivity to the 29/28-kDa proteins, suggesting that there is antigenic diversity (Chen et al., 1997). Recently molecular evidence has been generated to support antigenic diversity in the p28 gene from E. chaffeensis (Yu et al., 1998). A comparison of five E. chaffeensis isolates revealed that two isolates (Sapulpa and St. Vincent) were 100% identical, but three others (Arkansas, Jax, 91HE17) were divergent by as much as 13.4% at the amino acid level. The conservation of ECa28-1 suggests that E WO 00/32745 PCT/US99/28075 canis strains found in the United States may be genetically identical, and thus E. canis 28-kDa protein is an attractive vaccine candidate for canine ehrlichiosis in the United States. Further analysis of E. canis isolates outside the United States may provide information regarding the origin and evolution of E. canis. Conservation of the 28-kDa protein makes it an important potential candidate for reliable serodiagnosis of canine ehrlichiosis.

The role of multiple homologous genes is not known at this point; however, persistence of E.canis infections in dogs could conceivably be related to antigenic variation due to variable expression of homologous 28-kDa protein genes, thus enabling E canis to evade immune surveillance. Variation of msp-3 genes in A.

marginale is partially responsible for variation in the MSP-3 protein, resulting in persistent infections (Alleman et al., 1997). Studies to examine 28-kDa protein gene expression by E. canis in acutely and chronically infected dogs would provide insight into the role of the 28-kDa protein gene family in persistence of infection.

The following references were cited herein.

Alleman et al., (1997) Infect Immun65: 156-163.

Anderson et al., (1991) J Clin Microbiol 29: 2838-2842.

Anderson et al., (1992) Int J Syst Bacteriol 42: 299-302.

Brouqui et al., (1992) J Clin Microbiol 30: 1062-1066.

Chen et al., (1997) Clin Diag Lab Immunol 4: 731-735.

Chen et al., (1994) Am J Trop Med Hyg 50: 52-58.

Dawson et al., (1992) Am J Vet Res 53: 1322-1327.

Dawson et al., (1991) J Infect Dis 163: 564-567.

Donatien, et al., (1935) Bull Soc Pathol Exot 28: 418-9.

Ewing, (1963) J Am Vet Med Assoc 143: 503-6.

Groves et al., (1975) Am J Vet Res 36: 937-940.

Harrus et al., (1998) J Clin Microbiol 36: 73-76.

WO 00/32745 PCT/US99/28075 Jameson et al., (1988) CABIOS 4: 181-186.

Jongejan et al., (1993) Rev Elev Med Vet Pays Trop 46: 145-152.

McBride et al., (1996) J Vet Diag Invest 8: 441-447.

McBride, et (1999) Clin Diagn Lab Immunol.; (In press).

McClure, (1985) Ann Rev Biochem 54: 171-204.

McGeoch D.J. (1985) Virus Res 3: 271-286.

Nyindo et al., (1991) Am J Vet Res 52: 1225-1230.

Nyindo, et al., (1971) Am J Vet Res 32: 1651-58.

Ohashi, et al., (1998) Infect Immun 66: 132-9.

Ohashi, et al., (1998) J Clin Microb 36: 2671-80 Reddy, et al., (1998) Biochem Biophys Res Comm 247: 636-43.

Rikihisa, et al., (1994) J Clin Microbiol 32: 2107-12.

Rothbard et al., (1988) The EMBO J7: 93-100.

Sambrook et al., (1989) In Molecular Cloning: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Press.

Troy et al., (1990) Canine ehrlichiosis. In Infectious diseases of the dog and cat Green C.E. Philidelphia: W.B. Sauders Co.

von Heijne, (1986) Nucl Acids Res 14: 4683-90.

Walker, et al., (1970) J Am Vet Med Assoc 157: 43-55.

Weiss et al., (1975) Appl Microbiol 30: 456-463.

Yu, et al., (1997) Gene 184: 149-154.

Yu, et al., (1998) J. Clin. Microbiol. (In press).

Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. These patents and publications are herein incorporated by reference to the same extent as if each individual publication was individually indicated to be incorporated by reference.

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain WO 00/32745 PCT/US99/28075 the ends and advantages mentioned, as well as those inherent therein.

The present examples along with the methods, procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.

Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification, they are to be interpreted as specifying the presence of the stated features, integers, steps or components referred to, but not to preclude the presence or addition of one or more other feature, integer, step, component or group thereof.

0 0 EDITORIAL NOTE APPLICATION NUMBER 19234/00 The following Sequence Listing pages 1/24 to 24/24 are part of the description. The claims pages follow on pages "46" to "47".

WO 00/32745 PCTIUS99/28075 <110> <120> <130> <141> <150> <151> <160> <210> <211> <212> <213> <220> <223> <400> SEQUENCE LISTING Walker, David H.

McBride, Jere W.

Yu, Xue-Jie Homologous 28-Kilodalton Immunodorninant Protein Genes of Ehr-lichia canis and Uses Thereof D6 152 PCT 1999- 11-3 0 09/261,358 1999-03-03 33 1 1607

DNA

Ehrlichia canis nucleic acid sequence of ECa28-1 1 attttattta atc tc taatg gcacttccac ttgcaaaaaa ttccaagcat aacttctata cttctcagct aacatgattg actgttccaa tgcaggagc t tatcttatga gacgcgcaca agctgataaa ttgcaataaa ccttatatat tacaagtcct ttaatccgga ggtaatgagt aataagtgga ttaccaatct tatataatat ttttatacct aatatatata tattgttaat ttattttcac attcttataa caactgcatt atctttttct gatactatac ttagtggaaa gtatgtacca aaagaagaaa gcaaatcaac ggatggaagt ccaatactta actattcgtt cagatacgag atcggttact caatgggtgg agcattcgac gtaaaaagtc ggtactgcgc tctatctcat tttgtcttct taaaaaacga tgcatgttat gatataataa gcgcaggtat tggtactgat aaaatttcct accaaggaaa aacctctgtt ttcatcggtg ttagagatat tcctgcaata.

ccacaatttg caacagtaac attaaatttc ttctggcttg tattttaggt aatatcatta aagatggtaa agtgtctcac tgttggagtt agaataaaca aacaatccat cccaagaata c taatatcaa cacacatcgg agggttaatt atgacaaagt ttgatttcta actgggcatt ggcatttcca gtacctagta actaaatgtg tcttacaaaa tatctacttt gtaatatgaa atgtactcta catgggtggt attttggtag tttggattaa cgctgacttt ttctagggtt gaattcgaaa ttatcaaaat cagccatgga gacatatcac acctgtttct tgtttgaagc agttactcta caggatcata actcaactac tgtcactttg 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 SEQ 1/24 WO 00/32745 PCT[US99/28075 gtttagaact ttaaaaatga aaatagtggc tatcttctat agaaggtaat ccaatcttat tataccaaaa tttaatttat ttttcatagc tctttttctg tagtgcaaag aagaagagaa gacggagcaa tacaatg tggaggaaga tttaacttct aattttattg tctaaacttg tttttawtat tgctacatac aaaagaatgt agcaataaga gggggggggg gcttcccaag ttttttcycg ctatttatga atcctcacgg aaaacttatc ttcaaatatt ataatatatt aaatttctct tacaaaaatc tatatattct gacttgcttt tcttctgcac ttgtcactat taggttataa taawatgaat aagtgcattg atatcactaa tgtctttctt aatcaataca tgaagataat ataaatggta tatatgccaa gtgcctcaca ctttggcgta aaacacaaca actggagttt tcggattaaa cactaaagga tgcaagcwgc agccacacaw ttgccacata aaaaaaagaa ggaccaaatt cttaaacaac ttatttatta actagtattt ttctactatt tgcmaaagat acc tagcgta acttttacat ttttcagtta acaagattgg tagacccaag 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1607 <210> <211> <212> <213> <220> <223> <400> Met Asn Cys Met Tyr Ser Gly Asn Met Ser Val Ser Ser Thr Val Pro Ile Leu Ser Phe Arg Ile Gly Tyr 2 278

PRT

Ehrlichia canis amino acid sequence of ECa28-1 protein 2 Lys Lys Ile Leu Ile Thr Thr Ala Leu Ile Ser Leu 10 Ile Pro Ser Ile Ser Phe Ser Asp Thr Ile Gin Asp 25 Gly Gly Asn Phe Tyr Ile Ser Gly Lys Tyr Val Pro 40 His Phe Gly Ser Phe Ser Ala Lys Glu Giu Ser Lys 55 Gly Val Phe Gly Leu Lys His Asp Trp Asp Gly Ser 70 Lys Asri Lys His Ala Asp Phe Thr Val Pro Asn Tyr 85 Tyr Giu Asn Asn Pro Phe Leu Gly Phe Ala Gly Ala 100 105 Ser Met Gly Gly Pro Arg Ile Giu Phe Glu Ile Ser 110 115 120 SEQ 2/24 WO 00/32745 WO 0032745PCTIUS99/28075 Tyr Glu Ala Phe Asp Ala His Arg Met Glu Ala Asp Asp Ile Ser Leu Lys Val Pro Val Leu Ile Ser Met Gly Lys Leu Gly Phe Ile Giy Gly Asp Ile Pro Ala Pro Gin Phe Ala Giu Leu Gly Gly Asp Val Lys Ser Pro Asi Ile 125 130 Tyr Cys Ala Leu Ser His His 140 145 Lys Phe Val Phe Leu Lys Asn 155 160 Ala Ile Asn Ala Cys Tyr Asp 170 175 Ser Pro Tyr Ile Cys Ala Gly 185 190 Phe Giu Ala Thr Ser Pro Lys 200 205 Ile Ser Tyr Ser Ile Asn Pro 215 220 His Phe His Arg Ile Ile Giy 230 235 Ile Val Pro Ser Asn Ser Thr 245 250 Thr Val Thr Leu Asn Vai Cys 260 265 Arg Phe Asn Phe 275 Asn Tyr Thr Ser Giu Gly Ile Ile Ile Gly Ile Ser Glu Thr Asn Glu.

Thr Ile His Phe Gin Asn 135 Ala Ala 150 Leu Ile 165 Asn Asp 180 Thr Asp 195 Tyr Gin 210 Ser Val 225 Phe Arg 240 Ser Gly 255 Gly Leu 270 <210> <211> <212> <213> <220> <221> <223> <400> 3 849

DNA

Ehrlichia cani~s mat-peptide nucleic acid sequence of ECa28SA2 3 atgaattgta cttcctacct atggtaattt ggaatttttt cttaaaagaa ataattttac aaaaagtttt cacaataagt gcattgatat catccatata aatgtctcat actctaaccc agtatatggt aacagtatgt 100 ttacatatca ggaaagtaca tgccaagtgt tcctcatttt 150 cagctgaaga agagaaaaaa aagacaactg tagtatatgg 200 aactgggcag gagatgcaat atctagtcaa agtccagatg 250 cattcgaaat tactcattca agtatgcaag caacaagttt 300 SEQ 3/24 WO 00/32745 PCT[US99/28075 ttagggtttg agttgagatg ac aaaaacgg gatgatgaca aggattactt gcaaaaatat ttaattcaca gctagggttg tatattttca gtacctatta attaaatgta cagtagctat tcttatgaag tgcttacagg tgac tagtgc aacatatcat acctctctct tgtttgaaac gcctacttcg taaaattata actcagacga tgctactttg tggttactcg ataggcagtc caagaataga catttgatgt gaaaaatcca ggtgataatt tattgtgctt tatctcatca agatgatgcg aactgacaaa tttgtatatt taattaatga ttatgacaaa catatgttat gaaacagcaa ccttacatat gtgcaggtat tggtactgat tacacatcct aaaatttctt atcaaggaaa taagtgcaga gtcttcggtt tcttttggta aataataagt ttaaaaatgt tccagccatg gatagtagga ccacagtttg caacagtaac gattagaact tggatgtagg ttcaacttc 350 400 450 500 550 600 650 700 750 800 849 <210> <211> <212> <213> <220> <223> <400> 4 283

PRT

Ehrlichia canis amino acid sequence of ECa28SA2 protein 4 Met Asn Cys Ile Tyr Phe Asn Ser Met Ser Val Pro Lys Thr Thr Ala Ile Ser Tyr Ser Phe Ala Ile Gly Ser Tyr Giu Lys Lys Val Phe Thr Ile Ser Ala 10 Leu Pro Asn Val Ser Tyr Ser Asn 25 Tyr Gly Asn Phe Tyr Ile Ser Gly 40 His Phe Gly Ile Phe Ser Ala Giu 55 Val Val Tyr Giy Leu Lys Giu Asn 70 Ser Gin Ser Pro Asp Asp Asn Phe 85 Lys Tyr Ala Ser Asn Lys Phe Leu 100 Tyr Ser Ile Gly Ser Pro Arg Ile 110 115 Ala Phe Asp Val Lys Asn Pro Giy 125 130 Leu Ile Ser Pro Val Tyr Lys Tyr Met Giu Giu Lys Trp Ala Gly Thr Ile Arg Gly Phe Ala Giu Val Giu Asp Asn Tyr Ser Gly Pro Lys Asp Asn Val 105 Met 120 Lys 135 SEQ 4/24 WO 00/32745 WO 0032745PCTIUS99/28075 Asn Gly Ala Tyr Arg Tyr Cys Ala Leu Ser His 140 145 Asp Asp Asp Met Thr Ser Ala Thr Asp Lys Phe Asn Giu Gly Glu Thr Ala Gly Ile Gly Lys Ile Ser Ala Glu Ser Asn Asn Lys Asp Giu Ile 155 160 Leu Leu Asn Ile Ser Phe Met Thr 170 175 Ser Lys Asn Ile Pro Leu Ser Pro 185 190 Thr Asp Leu Ile His Met Phe Glu 200 205 Tyr Gin Gly Lys Leu Gly Leu Ala 215 220 Ser Val Ser Phe Giy Ile Tyr Phe 230 235 Phe Lys Asn Val Pro Ala Met Val 245 250 Val Gly Pro Gin Phe Ala Thr Val 260 265 Gin Asp Asp Ala 150 Val Tyr Leu Ile 165 Asn Ile Cys Tlyr 180 Tyr Ile Cys Ala 195 Thr Thr His Pro 210 Tyr Phe Val Ser 225 His Lys Ile Ile 240 Pro Ile Asn Ser 255 Thr Leu Asn Val 270 Cys Tyr Phe GiyLeu Giu Leu Gly Cys Arg Phe Asn Phe 275 280 <210> <211> <212> <213> <220> <221> <223> <400> 840

DNA

Ehrlichia canis mat-peptide nucleic acid sequence of ECa28SA3 atgaattgca ctatgctcca gtagcttcta gttttctcag aaaacatgat tattcacagt gggtttgcag tgaagttctg aaaaaattct tataacaact gcattaatgt agcatatctt tttctgatac tatacaagac catcagtgga aaatatgtac caagtgtttc ctaaagaaga aagaaactca actgttggag tggaatggag gtacaatatc taactcttct tcaaaattat tcgtttaaat acgaaaacaa gagctattgg ttattcaatg ggtggcccaa tacgagacat tcgatgtgaa aaatcagaac cattaatgta gataacactg acattttggt tttttggatt ccagaaaata cccattctta gaatagaact aataattata 100 150 200 250 300 350 400 SEQ 5/24 WO 00/32745 WO 0032745PCT/US99/2 8075 agaacggcgc agcatgtcct aattgactta gaatgccttt tccatgtttg attaggttat ttcacagagt agtggatcaa gtgtcacttt acacagatac ccgcaagtaa tcatttatga ttcaccttat aagctataaa agtataagtt cataggtaat atcttccaga ggcatagaac tgtgctttat ctcatcatag ttcagcaaca caaatttgtt ttcttaaaaa atgaagggtt taaatgcatg ctatgacata ataattgaag atttgtgcag gtgttggtac tgatgttgtt tcctaaaatt tcttaccaag gaaaactagg cagaagcctc tgtttttatc ggtggacact gaatttagag acatccctgc tatggttcct aaaccaattt gcaatagtaa cactaaatgt ttggaggaag atttaacttc 450 500 550 600 650 700 750 800 840 <210> <211> <212> <213> <220> <223> <400> 6 280

PRT

Ehrlichia canis amino acid sequence of ECa28SA3 protein 6 Met Asn Cys Lys Met Tyr T-yr Ala Asp Asn Thr Gly Val Ser His Phe Thr Val Gly Val Ile Ser Asn Ser Ser Phe Lys Tyr Ile Gly Tyr Ser T'yr Giu Thr Phe Gly Ala His Arg Lys Ile Leu Ile Thr Thr Ala Leu Met Ser Leu 10 Pro Ser Ile Ser Phe Ser Asp Thr Ile Gin Asp 25 Ser Phe Tyr Ile Ser Gly Lys Tyr Val Pro Ser 40 Gly Val Phe Ser Ala Lys Glu. Giu Arg Asn Ser 55 Phe Gly Leu Lys His Asp Trp, Asn Gly Gly Thr 70 Ser Pro Giu Asn Ile Phe Thr Val Gin Asn Tyr 85 Glu Asn Asn Pro Phe Leu Gly Phe Ala Gly Ala 100 105 Met Gly Gly Pro Arg Ie Glu Leu Glu Val Leu 110 115 120 Asp Val Lys Asn Gin Asn Asn. Asn Tyr Lys Asn 125 130 135 Tyr Cys Ala Leu Ser His His Ser Ser Ala Thr 140 145 150 SEQ 6/24 WO 00/32745 PCT/US99/28075 Ser Met Ser Ser Ala Ser Asn Lys Phe Val Phe Leu Lys Asn Glu Gly Ile Gly Ser Ala Glu Pro Gly Leu Ile Ile Glu Thr Asp Tyr Gin Ser Val Phe Arg Glu Asn Ile Glu Asp Gly Val Gly Phe Asp Gin Leu 155 Leu 170 Met 185 Val 200 Lys 215 Ile 230 Ile 245 Phe 260 Gly 275 7 133 Ser Phe Pro Phe Ser Met Leu Gly Gly Gly Pro Ala Ala Ile Gly Arg 160 Met Ile Asn 175 Ser Pro Tyr 190 Phe Glu Ala 205 Leu Gly Tyr 220 His Phe His 235 Met Val Pro 250 Val Thr Leu 265 Phe Asn Phe 280 Ala Cys Ile Cys Ile Asn Ser Ile Arg Val Ser Gly Asn Val 165 Tyr Asp Ile 180 Ala Gly Val 195 Pro Lys Ile 210 Ser Ser Glu 225 Ile Gly Asn 240 Ser Asn Leu 255 Cys His Phe 270 <210> <211> <212> <213> <220> <223> <400> Met Asn Cys Ile Tyr Phe Asn Ser Met Ser Val Pro Lys Thr Thr Ala Ile Ser

PRT

Ehrlichia canis partial amino acid sequence of ECa28SA2 protein 7 Lys Leu Tyr His Val Lys Pro Gly Phe Val Val Phe Thr Ile Ser Ala Leu Ile Ser Ser 10 Asn Val Ser Tyr Ser Asn Pro Val Tyr Gly 25 Asn Phe Tyr Ile Ser Gly Lys Tyr Met Pro 40 Gly Ile Phe Ser Ala Glu Glu Glu Lys Lys 55 Tyr Gly Leu Lys Glu Asn Trp Ala Gly Asp 70 Ser Gin Ser Pro Asp Asp Asn Phe Thr Ile Arg Asn 85 SEQ 7/24 WO 00/32745 PCT/US99/28075 Tyr Ser Phe Lys Tyr Ala Ser Asn Lys Phe Leu Gly Phe Ala Val Ala Ser Ile Gly Tyr Glu 100 105 Tyr Ser Ile Gly Ser Pro Arg Ile Glu Val Glu Met 110 115 120 Ala Phe Asp Val Lys Asn Gin Gly Asn Asn 125 130 Met Thr Ser Ala Thr Ile Ser Ile His Asp Ser Phe <210> <211> <212> <213> <220> <223> <400> Lys Tyr Ser Phe Thr Ile Ser His Lys Val Asn Asn Phe Lys Gly Tyr Glu Ile Ser His Asp Gly Val Leu Lys Thr His Phe Leu Asn Tyr Ser Phe Lys Asn Leu Lys Thr Phe Thr His Phe Ile Pro Asn Phe Tyr Ile Gly Ile Phe Ser Val Gly Leu Asp Asp Thr Ala Lys Lys Asn Asn Pro Ile Gly Asn Ser 110 Asp Thr Lys Asn 125 Tyr Cys Ala Leu 140 Ser Gly Asp Trp 155 Lys Asn Glu Gly 170 Val Phe Ser Ala Gin Ser Phe Arg Pro Ser Tyr Leu Thr Ala Leu 10 Tyr Ser Pro 25 Gly Lys Tyr 40 Lys Glu Glu 55 Arg Leu Ser 70 Leu Lys Val 85 Leu Gly Phe 100 Ile Glu Leu 115 Gly Asn Asn 130 His Gly Ser 145 Thr Ala Lys 160 Leu Asp Val 175 8 287

PRT

Ehrlichia canis amino acid sequence of ECa28SAl protien 8 Val Leu Leu Ala Arg Ala Met Pro Thr Gin Ser Phe His Asn Ile Gin Asn Tyr Ala Gly Ala 105 Glu Val Ser 120 Tyr Leu Asn 135 His Ile Cys 150 Thr Asp Lys 165 Ser Phe Met 180 SEQ 8/24 WO 00/32745 Leu Asn Ala Cys Pro Tyr Ile Cys Giu Thr Thr Gin Asn Tyr Thr Ile Phe His Lys Val Leu Pro Asp Gly Thr Leu. Asp Val Phe Phe Tyr Asp 185 Ala Gly 200 Asn Lys 215 Asn Ser 230 Ile Gly 245 Ser Asn 260 Cys His 275 Ile Thr Thr Giu.

190 Ile Gly Thr Asp 205 Ile Ser Tyr Gin 220 Arg Val Ser Val 235 Asn Giu Phe Lys 250 Ile Lys Val Gin 265 Phe Gly Leu Glu.

280 Lys Leu Gly Phe Gly Gin Ile PCT1US99/28075 Met Pro Phe Ser 195 Ile Ser Met Phe 210 Lys Leu Gly Leu.

225 Ala Gly Gly His 240 Ile Pro Thr Leu.

255 Ser Ala Thr Val 270 Gly Ser Arg Phe 285 Met Ile Gly Ala Thr Ile Ser <210> <211> <212> <213> <220> <223> <400> Asn Tyr Ser Ser Ile Asn Ser His Val Giy Ser Asn Phe Lys Lys Leu Gly Phe Val Ser Tyr 9 281

PRT

Ehrlichia chafifeensis amino acid sequence of chaffeensis P28 9 Lys Val Phe Ile Thr Ser Ala Leu Ile Ser Leu 10 Pro Giy Val Ser Phe Ser Asp Pro Ala Gly Ser 25 Asn Phe Tyr Ile Ser Gly Lys Tyr Met Pro Ser 40 Gly Val Phe Ser Ala Lys Glu Glu Arg Asn Thr 55 Phe Gly Leu Lys Gin Asn Trp Asp Gly Ser Ala 70 Ser Pro Asn Asp Val Phe Thr Val Ser Asn Tyr 85 Giu Asn Asn Pro Phe Leu Giy Phe Ala Gly Ala 100 105 SEQ 9/24 WO 00/32745 WO 0032745PCTIUS99/28075 Ile Giy Tyr Giu Giu Ala Met Ser Leu Leu Gly Giu Thr Asp Tyr Gin Ser Val Phe Arg Giy Lys Phe Gily Tyr Thr His Ser Asp Gly Leu Giy Phe Asp Ser Phe Arg Ala Ile Ile Val Lys Ile Ile Met 110 Asp 125 Tyr 140 Ser 155 Ser 170 Pro 185 Ser 200 Leu 215 Giy 230 Pro 245 Tyr 260 Leu 275 Asp Val Cys Asn Phe Phe Met Gly Giy Thr Pro Gly Gly Lys Ala Asn Met Ser Phe Leu His Ile Ala Gly Pro Asn Leu Phe Leu Pro Giu Ser Phe Ile Ile Arg Arg Gin Ser Val Asn Tyr Ala Tyr His Pro Val Phe Ile 115 Giy 130 His 145 Phe 160 Ala 175 Ile 190 Thr 205 Ser 220 Lys 235 Thr 250 Ile 265 Ala 280 Giu Asn Asn Leu Cys Cys Asn Ile Val Gly Leu Phe Leu Giu Val Asn Tyr Lys Ser Ala Ala Lys Asn Giu Tyr Asp Vai Ala Gly Ile Pro Lys Ile Ser Pro Giu Ile Giy Asn Ser Thr Leu Asp Val Cys Ser 120 Asn 135 Asp 150 Gly 165 Vai 180 Gly 195 Ser 210 Ala 225 Giu 240 Ala 255 His 270 Gly Asn Ile Giu <210> <211> <212> <213> <220> <223> <400> Met Asn Tyr Met Ser Ile 283

PRT

Ehrlichia chaffeensis amino acid sequence of E. chafffeensis OMP-lB Lys Lys Ile Phe Vai Ser Ser Ala Leu Ile Ser Leu 10 Leu Pro Tyr Gin Ser Phe Ala Asp Pro Val Thr Ser 25 SEQ 10/24 WO 00/32745 Asn Asp Thr Gly Val Lys Tyr Asn Glu Glu Ala Pro Phe Gly Leu Lys Asn Arg Thr Asp Gly Phe Ser Gly Glu Leu Glu Ala Asn Asn Asp Thr Ser Arg Glu Asp Asn Glu Gly Ile Asp Ile Thr Ala Gly Val Gly Ala Lys Phe Ser Tyr Pro Glu Val Ser Gly Asn Asn Phe Glu Gly Ala Pro Gly Tyr Phe Gly Ile Asn Asp Pro Ser Ile Ile Asn Gly Lys Asp Gly Pro Ala Leu Ser Ile Gly 110 Ala Tyr Gin 125 Asn Ser Gly 140 Ala Ile Ala 155 Thr Phe Met 170 Glu Gly Val 185 Asp Leu Ile 200 Gin Gly Lys 215 Ala Phe Ile 230 Asn Lys Ile 245 Gin Thr Thr 260 Gly Glu Val 275 Ser Ser Asn Asp Glu Tyr Lys Asp Asp Ser Pro Asn Ile .Gly Pro Ser Gly Arg Glu 40 His Phe 55 Thr Ser 70 Ile Ala 85 Phe Gin 100 Ala Met 115 Phe Asp 130 Tyr Tyr 145 Lys Lys 160 Leu Met 175 Phe Ile 190 Val Phe 205 Gly Ile 220 Gly Tyr 235 Val Ile 250 Ala Leu 265 Val Arg 280 PCT/US99/28075 Gly Phe Tyr Ile Ser Arg Lys Phe Ser Ala Ile Thr Lys Lys Val Gin Ser Ala Asn Phe Asn Asn Leu Ile Ser 105 Asp Gly Pro Arg Ile 120 Ala Lys Asn Pro Asp 135 Lys Tyr Phe Gly Leu 150 Tyr Val Val Leu Lys 165 Val Asn Thr Cys Tyr 180 Pro Tyr Ala Cys Ala 195 Lys Asp Phe Asn Leu 210 Ser Tyr Pro Ile Thr 225 Tyr His Gly Val Ile 240 Thr Pro Val Val Leu 255 Val Thr Ile Asp Thr 270 Phe Thr Phe <210> <211> 11 280 SEQ 11/24 WO 00/32745 PCT/US99/28075 <212> <213> <220> <223> <400> Met Asn Cys Met Ser Phe Asp Ser Val Ser Ala Ser Pro Thr Val Ser Ala Ser Ser Phe Lys Ile Gly Tyr Tyr Glu Thr Asp Ala His Asn Ala Thr Leu Asp Ile Glu Gly Ile Asp Leu Ile Gin Gly Lys

PRT

Ehrlichia chaffeensis amino acid sequence of E. chaffeensis OMP-1C 11 Lys Leu Ser His Ala Ser Tyr Ser Phe Arg Ala Ser Pro Ser Leu Lys Phe Pro Gly Gly Asn Phe Gly Leu Tyr His Ala Glu Asn Met Gly 110 Asp Val 125 Tyr Cys 140 Ser His 155 Leu Met 170 Phe Ser 185 Met Phe 200 Gly Leu 215 Phe Ile Ile Leu Phe Tyr Val Phe Gly Leu Asp Ala Asn Pro Gly Pro Lys Asn Ala Leu Tyr Val Leu Asn Pro Tyr Glu Ala Ser Tyr Thr Thr 10 Leu Ser 25 Ile Ser 40 Ser Ala 55 Lys Gin 70 Asp Phe 85 Phe Leu 100 Arg Ile 115 Gin Gly 130 Asp Arg 145 Leu Leu 160 Ala Cys 175 Ile Cys 190 Ile Asn 205 Ser Ile Ala Glu Gly Lys Asp Asn Gly Glu Gly Lys Lys Tyr Ala Pro Asn Leu Ala Leu Pro Pro Val Gin Asp Lys Tyr Met Pro Glu Glu Lys Asn Trp Asn Gly Val Asn Lys Gly Tyr Phe Ala Gly Ala 105 Phe Glu Val Ser 120 Asn Tyr Lys Asn 135 Ala Ser Ser Thr 150 Asn Glu Gly Leu 165 Asp Val Val Ser 180 Gly Val Gly Thr 195 Lys Ile Ser Tyr 210 Pro Glu Ala Ser 220 Val Phe Val Gly Gly His Phe His Lys Val 225 Ala Gly Asn Glu Phe 240 230 235 SEQ 12/24 WO 00/32745 PCT/US99/28075 Arg Asp Ile Ala Thr Pro Gly Val Glu Ser Thr Leu Lys Ala Phe Ala Thr Pro Ser Ser Ala 245 250 255 Asp Leu Ala Thr Val Thr Leu Ser Val Cys His Phe 260 265 270 Leu Gly Gly Arg Phe Asn Phe 275 280 12 286

PRT

Ehrlichia chaffeensis amino acid sequence of E. chaffeensis OMP-1D 12 Met Met Asp Ser Thr Val Tyr Ala Ser Asn Glu <210> <211> <212> <213> <220> <223> <400> Asn Cys Ser Phe Asn Ile Ala Ser Thr Val Ile Ser Ser Phe Ile Gly Tyr Glu Glu Ala Thr Gin Glu Leu Ser His Gly Arg Lys Tyr Ala His Ile Lys Phe Phe Ile Pro Gly Ile Ser Gly Asn Phe Tyr Phe Gly Val Phe Val Phe Gly Ile Thr Thr Leu Ser Tyr Glu Asn Asn Ser Met Asp Gly 110 Phe Asp Val Lys 125 Arg Tyr Tyr Ala 140 Asp Gly Ala Gly 155 Thr Thr Ala Leu Thr Leu Leu 10 Leu Ser Asp Pro Val Gin Asp 25 Ile Ser Gly Lys Tyr Met Pro 40 Ser Ala Lys Glu Glu Arg Asn 55 Glu Gin Asp Trp Asp Arg Cys 70 Asp Ile Phe Thr Val Pro Asn 85 Leu Phe Ser Gly Phe Ala Gly 100 105 Pro Arg Ile Glu Leu Glu Val 115 120 Asn Gin Gly Asn Asn Tyr Lys 130 135 Leu Ser His Leu Leu Gly Thr 145 150 Ser Ala Ser Val Phe Leu Ile 160 165 SEQ 13/24 WO 00/32745 Asn Glu Gly Asp Val Ile Gly Ile Gly Lys Ile Ser Pro Glu Ala Gly Asn Glu Ala Leu Ala Val Phe Tyr Leu <210> <211> <212> <213> <220> <223> <400> Met Asn Cys Met Ser Phe Asp Asn Ile Ser Ala Ser Pro Thr Val Ser Ser Ser Leu Leu 170 Ser Glu 185 Ile Asp 200 Tyr Gin 215 Ser Val 230 Phe Arg 245 Gly Lys 260 Phe Gly 275 PCT/US99/28075 Asp Lys Ser Phe Met Leu Asn Ala Cys Tyr 175 180 Gly Ile Pro Phe Ser Pro Tyr Ile Cys Ala 190 195 Leu Val Ser Met Phe Glu Ala Ile Asn Pro 205 210 Gly Lys Leu Gly Leu Ser Tyr Pro Ile Ser 220 225 Phe Ile Gly Gly His Phe His Lys Val Ile 235 240 Asp Ile Pro Thr Met Ile Pro Ser Glu Ser 250 255 Gly Asn Tyr Pro Ala Ile Val Thr Leu Asp 265 270 Ile Glu Leu Gly Gly Arg Phe Asn Phe Gin 280 285 13 278

PRT

Ehrlichia chaffeensis amino acid sequence of E. chaffeensis OMP-1E 13 Lys Lys Phe Phe Ile Thr Thr Ala Leu Val Ser Leu 10 Leu Pro Gly Ile Ser Phe Ser Asp Pro Val Gin Gly 25 Ser Gly Asn Phe Tyr Val Ser Gly Lys Tyr Met Pro 40 His Phe Gly Met Phe Ser Ala Lys Glu Glu Lys Asn 55 Ala Leu Tyr Gly Leu Lys Gin Asp Trp Glu Gly Ile 70 Ser His Asn Asp Asn His Phe Asn Asn Lys Gly Tyr 85 SEQ 14/24 WO 00/32745 PCT/US99/28075 Ser Ile Tyr Asp Ile Leu Glu Asp Gin Val Arg Pro Glu Phe Gly Glu Ala Pro Asp Ser Leu Gly Phe Asp Asp Leu Lys Tyr Thr His Lys Ile Ile Ile Lys Ile Ile Leu Gly Tyr Glu Asn Asn Pro Phe Leu Gly Phe Ala Gly Ala 100 105 Ser Phe Arg Thr Ser Pro Ser Leu Gly Pro Ala Gly Met 110 Asp 125 Tyr 140 Ser 155 Phe 170 Leu 185 Met 200 Gly 215 Gly 230 Thr 245 Ile 260 Arg 275 Gly Val Cys Lys Met Ser Phe Leu His Leu Val Phe Gly Pro Lys Asn Ala Leu Tyr Val Leu Asn Pro Tyr Glu Ala Ser Tyr Phe His Lys Ala Thr Leu Asn Phe Arg Val 115 Gin Gly 130 Gly Gin 145 Leu Leu 160 Ala Cys 175 Ile Cys 190 Thr Asn 205 Ser Ile 220 Lys Val 235 Phe Val 250 Ser Val 265 Glu Phe Asn Asn Gin Asp Lys Ser Tyr Asp Ala Gly Pro Lys Asn Pro Ile Gly Thr Ser Cys His Glu Tyr Asn Glu Ile Val Ile Glu Asn Ser Phe Val Lys Ser Gly Ile Gly Ser Ala Glu Ala Gly Ser 120 Asn 135 Gly 150 Leu 165 Asn 180 Thr 195 Tyr 210 Ser 225 Phe 240 Thr 255 Ile 270 <210> <211> <212> <213> <220> <223> <400> Met Asn Cys 280

PRT

Ehrlichia chaffeensis amino acid sequence of E. chaffeensis OMP-1F 14 Lys Lys Phe Phe Ile Thr Thr Thr Leu Val Ser Leu 10 SEQ 15/24 WO 00/32745 Met Ser Phe Leu PCT/US99/28075 Pro Gly Ile Ser Phe Ser Asp Ala Val Gin Asn 25 tn Asp Ser Thr Thr Tyr Ala Ser Asn Lys Gly Ile Gly Ser Ala Glu Thr Asn Val Val Ser Thr Thr Ile Ser Ser Phe Val Gly Tyr Glu Asp Ala Leu Ser Leu Leu Ser Glu Thr Asp Tyr Gin Ser Val Phe Arg Gly Asn Gly Gly Asn His Phe Gly Gly Val Phe Lys Asn Ser Lys Tyr Glu Tyr Leu Met 110 Thr Phe Asp 125 His Lys Tyr 140 Asn Ala Gly 155 Asp Ile Ser 170 Gly Ile Pro 185 Leu Ile Ser 200 Gly Lys Leu 215 Phe Val Gly 230 Asp Ile Pro 245 His Phe Thr 260 Phe Val Gly Pro Asn Asn Val Tyr Asp Leu Phe Met Gly Gly Ala Ile Tyr Phe Leu Glu Asn Gly Lys Ala Lys Met Ser Phe Leu His Met Val Ile Ser Lys Asn Pro Pro Asn Leu Phe Leu Pro Glu Ser Phe Ile Thr Ser 40 Ala 55 Gin 70 Thr 85 Phe 100 Arg 115 Gin 130 Thr 145 Val 160 Asn 175 Tyr 190 Ala 205 Tyr 220 His 235 Pro 250 Leu 265 Gly Lys Asp Phe Leu Ile Gly His Phe Ala Ile Ile Ser Lys Ser Ser Lys Tyr Val Pro Gin Glu Arg Asn Trp Asp Gly Ser Asn Val Pro Asn Gly Phe Ala Gly 105 Glu Leu Glu Met 120 Asn Asn Tyr Lys 135 Asn Ser Gly Gly 150 Leu Lys Asn Glu 165 Cys Tyr Asp Val 180 Cys Ala Gly Val 195 Asn Pro Lys Ile 210 Ile Ser Pro Glu 225 Val Ile Gly Asn 240 Thr Ser Thr Leu 255 Val Cys His Phe 270 Gly Val Glu Leu Gly Gly Arg Phe Asn Phe 275 280 SEQ 16/24 WO 00/32745 PCT/US99/28075 <210> <211> <212> <213> <220> <223> <400> Met Asn Cys Val Ser Phe Giu Asn Asn Pro Thr Ala Arg Asp Thr Vai Lys Thr Asp Tyr Ser Gly Ala Val Vai Ser Tyr Lys Asn Asp Ser Thr Ala Leu Thr Asp Leu Asp Gly Thr Asp Leu Tyr Gin Gly 284

PRT

Cowdria ruminantium amino acid sequence of C. ruminant lum MAP-i Lys Leu Pro Ser Lys Pro Phe Giy Glu Ala Giy Ile Met Val Lys Lys Pro Val His Ala Ser Lys Tyr 110 Thr 125 His 140 Ala 155 Ser 170 Pro 185 Ser 200 Leu 215 Ile Gly Gly Phe Vai Gly Ser Phe Met Thr Leu Val Val Gly Phe Ile Thr Ser 10 Val Ser Phe Ser 25 Ser Val Tyr Ile 40 Gly Lys Met Ser 55 Phe Gly Leu Lys 70 Asn Thr Asn Ser 85 Glu Asn Asn Pro 100 Met Asn Gly Pro 115 Asp Val Arg Asn 130 Tyr Cys Ala Leu 145 Thr Ser Val Met 160 Met Leu Asn Ala 175 Ser Pro Tyr Val 190 Ile Asn Ala Thr 205 Ile Ser Tyr Ser 220 Thr Asp Ser Ile Lys Ile Phe Arg Pro Asp Val Cys Cys Asn Ile Leu Val Ala Lys Asp Phe Leu Ile Giy Thr Lys Tyr Ala Pro Asn Ile Ile Lys Glu Trp Thr Gly Glu Gly Ala Asn Asp Gly Lys Pro Ser Gin Tyr Asp Asp Giu Phe Phe Asn Ser Glu Ile Ile Leu Glu Leu Glu Met Ser Gly Lys Ala 105 Glu 120 Tyr 135 Ser 150 Asn 165 Met 180 Gly 195 Ser 210 Ala 225 SEQ 17/24 WO 00/32745 PCT/US99/28075 Ser Ile Phe Ile Gly Gly His Phe His Arg Val Ile Gly Asn Glu 230 235 240 Phe Lys Asp Ile Ala Thr Ser Lys Val Phe Thr Ser Ser Gly Asn 245 250 255 Ala Ser Ser Ala Val Ser Pro Gly Phe Ala Ser Ala Ile Leu Asp 260 265 270 Val Cys His Phe Gly Ile Glu Ile Gly Gly Arg Phe Val Phe 275 280 <210> 16 <211> <212> DNA <213> artificial sequence <220> <221> primer_bind <222> nucleotides 313-332 of C. ruminantium MAP-1, also nucleotides 307-326 of E. chaffeensis P28 <223> forward primer 793 for PCR <400> 16 gcaggagctg ttggttactc <210> 17 <211> 21 <212> DNA <213> artificial sequence <220> <221> primer_bind <222> nucleotides 823-843 of C. ruminantium MAP-1, also nucleotides 814-834 of E. chaffeensis P28 <223> reverse primer 1330 for PCR <400> 17 ccttcctcca agttctatgc c 21 <210> 18 <211> 24 <212> DNA SEQ 18/24 WO 00/32745 PCT/US99/28075 <213> artificial sequence <220> <221> primerbind <223> primer 46f, specific for ECa28SA2 gene <400> 18 atatacttcc tacctaatgt ctca 24 <210> 19 <211> <212> DNA <213> artificial sequence <220> <221> primer_bind <223> primer used for sequencing 28-kDa protein genes in E. canis <400> 19 agtgcagagt cttcggtttc <210> <211> 18 <212> DNA <213> artificial sequence <220> <221> primer_bind <223> primer used for sequencing 28-kDa protein genes in E. canis <400> gttacttgcg gaggacat 18 <210> 21 <211> 24 <212> DNA <213> artificial sequence <220> <221> primer_band <222> nucleotides 687-710 of ECa28-1 SEQ 19/24 WO 00/32745 PCT/US99/28075 <223> primer 394 for PCR <400> 21 gcatttccac aggatcatag gtaa 24 <210> 22 <211> 24 <212> DNA <213> artificial sequence <220> <221> primer_band <222> nucleotides 710-687 of ECa28-1 <223> primer 394C for PCR <400> 22 ttacctatga tcctgtggaa atgc 24 <210> 23 <211> <212> DNA <213> artificial sequence <220> <221> primerbind <223> primer 793C which anneals to a region with Eca28-1, used to amplify the intergenic region between gene ECa28SA3 and ECa28-1 <400> 23 gagtaaccaa cagctcctgc <210> 24 <211> 24 <212> DNA <213> artificial sequence <220> <221> primer_band <222> <223> primer EC280M-F complementary to noncoding regions adjacent to the open reading frame SEQ 20/24 WO 00/32745 PCT[US99/28075 of ECa28-1 <400> tctactttgc acttccacta ttgt <210> <211> <212> <213> <220> <221> <223> <400> 24

DNA

artificial sequence primer_band primer EC280M-R complementary to noncoding regions adjacent to the open reading frame of ECa28-1 attcttttgc cactattttt cttt <210> <211> <212> <213> <220> <221> <223> <400> 26

DNA

artificial sequence primer_bind primer ECaSA3-2 corresponding to regions within ECa28SA3,used to amplify the intergenic region NC3 between gene ECa28SA3 and ECa28-1 26 ctaggattag gttatagtat aagtt <210> <211> <212> <213> <220> <221> <223> 27 23

PRT

Ehrlichia canis

PEPTIDE

a predicted N-terminal signal peptide of ECa28-1 SEQ 21/24 WO 00/32745 PCTIUS99/28075 Met Met <400> Asn Cys Tyr Tyr <210> <211> <212> <213> <220> <223> <400> Met Asn Tyr Ile Ser Ser <210> <211> <212> <213> <220> <223> <400> Met Asn Cys Met Tyr Ser <210> <211> and ECa28SA3 27 Lys Lys Ile Leu Ile Thr Thr Ala Leu Met Ser Leu 10 Ala Pro Ser Ile Ser 28

PRT

Ehrlichia chaffeensis amino acid sequence of N-terminal signal peptide of E. chaffeensis P28 28 Lys Lys Ile Leu Ile Thr Ser Ala Leu Ile Ser Leu 10 Leu Pro Gly Val Ser Phe Ser 29 26

PRT

Ehrlichia canis amino acid sequence of putative cleavage site of ECa2 8-1 29 Lys Lys Ile Leu Ile Thr Thr Ala Leu Ile Ser Leu 10 Ile Pro Ser Ile Ser Ser Phe Ser 299 SEQ 22/24 WO 00/32745 PTU9/87 PCTfUS99/28075 <212> <213> <220> <223> <400> taatacttct ttataaacgc tagaaaagtc ataaatgtta aaaagccact t ttgtat tt t <210> <211> <212> <213> <220> <223> <400> taatttcgtg ctgtatacaa tacaagtt La tttacaatga atgctttgtt actcactagt atacttccac

DNA.

Ehrlichia canis nucleic acid sequence of intergenic noncoding region 1 (28NC1) attgtacatg ttaaaaatag tactagtttg cttctgtggt aagagagaaa tagttagtaa taaattagaa agttaaatat 100 atatgttttt cattgtcatt gatactcaac taaaagtagt 150 cttattaata attttacgta gtatattaaa tttcccttac 200 agtattttat actaaaagct atactttggc ttgtatttaa 250 tactactgtt aatttacttt cactgtttct ggtgtaaat 299 31 345

DNA

Ehrlichia canis nucleic acid sequence of intergenic noncoding region 2 (28NC2) 31 gtacacatat gagaaaaaat ccaagcttat aatgtacact tattaatact aatttatact tattgttaat cacgaagcta aaattgtttt agtagtgaaa attacctaac tctcacaaaa cttcttgtgt tagcttcact actgtagagt ctacataata tgttaaattt agaatatata ttctgacttg ttattttcac tattttaggt tttatctctg aatatgacag cttttatctc gtgtttatca ttcttacaaa tatttgcttt gtaat 100 150 200 250 300 345 <210> <211> <212> <213> <220> <223> <400> 32 345

DNA

Ehrlichia canis nucleic acid sequence of intergenic noncoding region 3 (28NC3) 32 SEQ 23/24 WO 00/32745 PCTIUS99/28075 LgaLttttatt ttgctacata gagggggggg tatattcaaa ttttatttat atctctaatg gcacttccac gttgccacat caaaaaaaag ggggactaaa tagcacaact taccaatcct ttttaact tattgttaat attaaaaatg aaaaatagtg tttaccttct caatgcttcc tatataatat aaLa La La a ttattttcac atctaaactt gtttLtatta gcaaaagaat gtagcaataa attcttctaa tattctttac aggaaaatat gtttctaata attaaatttc tcttacaaaa ttctggcttg LaLLtactLL LatLLtaggL gtaaL 100 150 200 250 300 345 <210> <211> <212> <213> <220> <223> <400> 33 355

DNA

Ehrlichia canis nucleic acid sequence of intergenic noncoding region 4 (28NC4) 33 taaLLLtaLL Ltgc Lacata aggggggggg gctatttatg cttcaaatat Ltacaaaaat ttcttctgca ataaw gttgccacat aLtaaaaatg caaaaaaaga aaaatagtgg gggaccaaat LtatcLtcta acttaaacaa cagaaggtaa tLtaLLtaLL accaatctta cactagtatt ttataccaaa cLtctactaL Lttaattta atctaaactt gLLLLtawta caaaagaatg Lagcaataag 100 LgcLtcccaa gLLLLLtcyc 150 Latcctcacg gaaaacLtat 200 tataatataL LaaaLLtctc 250 atatataLtc tgacttgctt 300 LLtgtcacta LtaggLtata 350 355 SEQ 24/24

Claims (7)

1. An isolated DNA sequence encoding a 30-kilodalton protein of Ehrlichia canis, wherein said protein is immunoreactive with anti-Ehrlichia canis serum, and wherein said protein has an amino acid sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:6.

2. The DNA sequence of Claim 1, wherein said protein has an N-terminal signal sequence.

3. The DNA sequence of Claim 2, wherein said protein is post-translationally modified to a 28-kilodalton protein.

4. The DNA sequence of Claim 1, wherein said DNA has a sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID The DNA sequence of Claim 1, wherein said DNA is contained in a single locus of Ehrlichia canis.

6. The DNA sequences of Claim 5, wherein said locus is a multigene locus of 5.592 kb in length.

7. The DNA sequences of Claim 6, wherein said locus encoding homologous

28-kilodalton proteins of Ehrlichia canis. 8. The DNA sequences of Claim 7, wherein said homologous 28-kilodalton proteins of Ehrlichia canis are selected from the group consisting of ECa28SA1, ECa28SA2, ECa28SA3, ECa28-1 and ECa28-2. 9. A vector comprising the DNA sequences of any one of Claims 1 to 8. 10. The vector of Claim 9, wherein said vector is an expression vector capable S: of expressing a peptide or polypeptide encoded by the sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3 and SEQ ID NO:5 when said expression vector is introduced into a cell. 11. A recombinant protein comprising the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6. 12. The recombinant protein of Claim 11, wherein said amino acid sequence is S encoded by a nucleic acid segment comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3 and SEQ ID 13. A host cell comprising the nucleic acid segment selected from the group consisting of SEQ IDNO: 1, SEQ ID NO:3 and SEQ 14. A method of producing the recombinant protein of Claim 11, comprising the steps of: 46 24/04/03,swl2082spa,46 obtaining a vector that comprises an expression region comprising a sequence encoding the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6 operatively linked to a promoter; transfecting said vector into a cell; and culturing said cell under conditions effective for expression of said expression region. An antibody immunoreactive with an amino acid sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:6. 16. A method of inhibiting Ehrlichia canis infection in a subject comprising the steps of: identifying a subject suspected of being exposed to or infected with Ehrlichia canis; and administering a composition comprising a 28-kDa antigen of Ehrlichia canis in an amount effective to inhibit an Ehrlichia canis infection wherein said antigen is a recombinant protein and comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6. 17. The method of Claim 16, wherein said recombinant protein is encoded by a gene comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3 and SEQ ID 18. The method of Claim 16, wherein said recombinant protein is dispersed in a pharmaceutically acceptable carrier. 19. The isolated DNA sequence of Claim 1, or the method of Claim 14 or Claim 16 substantially as hereinbefore described in any one of the Examples. DATED this 24 th day of April, 2003 RESEARCH DEVELOPMENT FOUNDATION By Their Patent Attorneys: 30 CALLINAN LAWRIE 47 244/0/03,sw I 2082spa,47