AU772946B2 - Ehrlichia canis genes and vaccines - Google Patents

Description

W001/07625 PCT/US00/19763 Ehrlichia canis Genes and Vaccines FIELD OF THE INVENTION The invention pertains to the field of veterinary pathogens. More particularly, the present invention pertains to the sequence of specific genes of the bacterial canine pathogen Ehrlichia canis and the application of this technology to the development of a vaccine.

BACKGROUND OF THE INVENTION The present invention relates to the sequence of genes from the E. canis bacterium, and the development of a vaccine against this organism.

Ehrlichia canis canis) is a small gram-negative, obligately intracellular bacterium. This bacteria is the agent which causes canine monocytic ehrlichiosis (CME), a tick-borne disease which predominantly affects dogs. The most common carrier of E.

canis is the brown dog tick Rhipicephalus sanguineus. The disease was described originally in Algeria in 1935. It was subsequently recognized in the United States in 1962, but is now known throughout much of the world. Canine monocytic ehrlichiosis caused much concern during the Vietnam War, when 160 military dogs died from the E. canis infection. There is no vaccination currently available against E. canis. It is a life threatening disease that continues to be an important health concern for veterinarians and pet owners alike.

Canine monocytic ehrlichiosis is an infectious blood disease. A reduction in cellular blood elements is the primary characteristic of the disease. E. canis lives and reproduces in the white blood cells (leukocytes). It eventually affects the entire lymphatic system, and devastates multiple organs. By targeting the white blood cells, these cells die

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WO 01/07625 PCT/US00/19763 2 off rapidly. These dead blood cells migrate primarily to the spleen, which enlarges as a result. The bone marrow recognizes the loss of the white blood cells and works to form new, healthy cells. It sends out the cells prematurely, and these immature cells do not work properly. Often, these immature cells mimic those in leukemic patients, so the disease is misdiagnosed as leukemia. Canine monocytic ehrlichiosis may predispose dogs to various cancers.

There are three stages of canine monocytic ehrlichiosis. The first, acute stage mimics a mild viral infection. During the acute stage, most, if not all, of the damage is reversible and the animal is likely to recover. This is the stage where treatment is the most effective, stressing the need for early detection. Without treatment, however, the animal will progress into a subclinical (second) stage and/or to the chronic (final) stage. When the animal has reached the chronic stage, the bacterial organism has settled within the bone marrow. Many dogs in this stage suffer massive internal hemorrhage, or develop lethal complications such as sudden stroke, heart attack, renal failure, splenic rupture or liver failure.

E. canis can be cultured in vitro in a mammalian-derived cell line (DH82).

Continued maintenance of these cells is difficult because the cell culture must be supplemented with primary monocytes (white blood cells found in bone marrow) every two weeks. The cultures are very slow growing, and the culture media is expensive.

Data concerning the genes in the E. canis genome has concentrated primarily on the 16S rRNA gene. Previous work has sequenced this gene, which is a ubiquitous component of the members of the ehrlichia family, as well as the majority of organisms worldwide. The high sequence homology between this gene throughout the living world makes it a poor candidate for vaccine development. It is necessary to find other genes within this genome if hope for a vaccine against this deadly disease can ever be realized.

Sequencing of the 16S rRNA gene indicates that E. canis is closely related (98.2% homology) to E. chaffeensis, the novel etiologic agent of human ehrlichiosis. Western blots ofE. canis are similar when probed with antisera to E. canis, E. chaffeensis and E.

WO 01/07625 PCT/US00/19763 3 ewingi (another cause of human ehrlichiosis) indicating a close antigenic relationship between these three species (Chen et al., 1994).

The indirect fluorescent antibody test (IFA) has been developed for detecting canine monocytic ehrlichiosis. IFA detects the presence of antibodies against the invading organism in a dog's blood. Unfortunately, this test is not always accurate. Sometimes, dogs will test negative in the acute phase because their immune system is delayed in forming antibodies. Another false negative may occur if there is a low titer in the chronic stage. An additional drawback of this test is the cross-reactivity found. The anti E. canis polyclonal antibody positively reacts with E. chaffeensis, undermining the specificity of the test. An alternative test, the Giesma smear, has been used to locate the actual organism in a dog's blood. Unfortunately, despite appropriate staining techniques and intensive film examination, the organisms frequently can not be located. The fallibility of these tests makes it essential to provide better diagnostic tools for this disease.

Due to difficulties in the detection of a tick bite, early diagnosis of infection, the suppression of host defenses and the nature of persistent infection of the disease, an effective vaccine against E. canis is urgently needed for dogs.

SUMMARY OF THE INVENTION This invention discloses novel sequence data for E. canis genes. Specifically, a clone has been identified and sequenced. Four proteins termed ProA, ProB, ORF (an open reading frame with unknown function) and a cytochrome oxidase homolog, have been identified within this clone. In addition, a partial gene encoding a lipoprotein signal peptidase homolog has been discovered.

An embodiment of this invention includes the creation of a vaccine with this sequence and protein information. The proteins disclosed in this invention are extremely antigenic. Therefore, they have the potential to be extremely useful as a vaccine. The WO 01/07625 PCT/US00/19763 4 types of vaccine made available by this novel technology include a DNA vaccine, a recombinant vaccine, and a T cell epitope vaccine.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the three clones identified in the library screen.

DESCRIPTION OF THE PREFERRED EMBODIMENT E. canis causes a devastating canine disease. Currently, there is no vaccine available to prevent this disease. This invention provides the tools necessary to develop such a vaccine. More specifically, four genes have been identified from a genomic fragment of E. canis, named ProA, ProB, ORF and a cytochrome oxidase homolog. In addition, a partial gene coding for a lipoprotein signal peptidase homolog has been found.

Any of these proteins can be utilized in an embodiment of this invention to develop a vaccine.

Screening an E. canis library To identify genes in the E. canis genome, a genomic DNA expression library was constructed. An E. canis strain isolated from dogs with canine ehrlichiosis was grown in the dog cell line DH82 by a technique being known in the art, and incorporated by reference (Dawson et al., 1991; Rikihisa, 1992). The cells were harvested and the chromosomal DNA extracted as described by a technique known in the art (Chang et al., 1987; Chang et al., 1989a; Chang et al., 1989b; Chang et al., 1993a; Chang et al., 1993b).

To construct the library, 200 gLg of DNA was partially digested with Sau3A. DNA fragments from 3 to 8 kb were isolated and ligated to a plasmid, pHG165 (Stewart et al., 1986). The plasmids were transformed into E. coli TBI (Chang et al., 1987).

The library was screened with polyclonal antibodies against E. canis. Polyclonal antibodies were generated from dogs that had been bitten by a tick harboring E. canis.

WO 01/07625 PCT/US00/19763 The polyclonal antibodies were preabsorbed with the lysate of an E. coli host strain. The library was plated on petri plates at a density of 1,000 colony forming units. Colonies were transferred to nitrocellulose and each filter was probed with 1 ml of the preabsorbed polyclonal antibodies. Positive colonies were identified with a second antibody consisting of an alkaline phosphatase-conjugated goat anti-rabbit IgG (Kirkegaard and Perry Laboratories, Gaithersburg, MD), followed by color development with a substrate solution containing nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP). Positive clones were rescreened three times.

Three clones were isolated from this screening procedure (Figure The longest genomic fragment (pCH4) encodes four complete genes and one partial gene. It completely encodes the proteins ProA, ProB, ORF and a cytochrome oxidase homolog, as well as containing the partial sequence of a lipoprotein signal peptidase homolog. ProA and ProB are located on a single operon. Restriction endonuclease digestion mapping and DNA sequencing were done by techniques known in the art, and incorporated by reference (Chang et. al., 1987; Chang et. al., 1989a; Chang et. al., 1989b; Chang et. al., 1993a; Chang et. al., 1993b). Briefly, the DNA sequence was determined by automated DNA sequencing on the ABI PRISM Model 377 DNA system. The complete nucleotide sequences were determined on both strands by primer walking. The thermal cycling of the sequencing reactions utilized the Taq DyeDeoxy T M Terminator Cycle sequencing kit.

Databases were searched for homologous proteins through the use of the BLAST network service of the National Center for Biotechnology Information (NCBI) (Althchul et al., 1990; Gish et al., 1993).

Sequence Information The E. canis genes were sequenced. The cloned fragment contains 5,300 nucleotides, and codes for four proteins. There is also one partial gene at the carboxy terminus. SEQ. ID. NO. 1 is the entire nucleotide sequence. SEQ. ID. NO. 2 and 3 are the translation of nucleotides 12 through 533 from SEQ. ID. NO. 1 and code for a cytochrome oxidase homolog. Cytochrome oxidase is important in virulence, and therefore is a strong candidate for use in a vaccine. SEQ. ID. NO. 4 and 5 are the translation of nucleotides 939 through 2,252 from SEQ. ID. NO. 1 and code for ProA. SEQ. ID. NO. 6 and 7 are the WO 01/07625 PCT/USO/19763 6 translation ofnucleotides 2,258 through 3,664 from SEQ. ID. NO. 1 and code for ProB.

Preliminary evidence indicates that ProA and ProB are proteases. SEQ. ID. NO. 8 and 9 are the translation of nucleotides 4,121 through 4,795 from SEQ. ID. NO. 1 and code for ORF, a protein with unknown function. SEQ. ID. NO. 10 and 11 are the translation of the complementary sequence ofnucleotides 4,884 through 5,300 from SEQ. ID. NO. I and code for the partial sequence of a lipoprotein signal peptidase homolog. Lipoprotein signal peptidases are membrane proteins, and by nature may be less desirable for vaccine development. However, this protein is still worth pursuing in the creation of a vaccine.

Overexpression of ProA. ProB. ORF. cytochrome oxidase and the lipoprotein signal peptidase homolog The E. canis antigens are overexpressed in a T7 promoter plasmid. The pRSET vector allows high level expression in E. coli in the presence of T7 RNA polymerase, which has a strong affinity for the T7 promoter. After subcloning the antigen genes into the pRSET vector, the subclones are transformed into an F' E. coli JM109 strain. For maximum protein expression, the transformants are cultured to O.D. 600=0.3, exposed to IPTG (1 mM) for one hour and then transfected with M13/T7 bacteriophages at a multiplicity of infection (MOI) of 5-10 plaque forming units (pfu) per cell. Time course studies indicate that maximum induction is reached two hours after induction.

The pellet is harvested by centrifugation and the cells are resuspended in 6M Guanidinium (pH Cells are ruptured by French press and the total lysate is spun at 6000 rpm to separate cell debris by a technique known in the art, and hereby incorporated by reference (Chang et al., 1993c). Immobilized metal ion affinity chromatography (IMIAC) is used to purify each of the proteins under denaturing conditions as described by the manufacturer (Invitrogen, San Diego, CA). The protein samples are separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized after staining with coomassie blue.

Vaccine Development Prior to the present invention, no vaccine against E. canis had been developed. E.

canis is endemic in dogs and closely related canidae in many parts of the world. Dogs in WO 01/07625 PCTIUS00/19763 7 North America are also increasingly at risk and the application of the present invention can potentially save the lives of thousands of dogs each year. An E. canis vaccine that can elicit cell-mediated immunity against this tick-bore disease of dogs is desperately needed.

DNA Vaccine A DNA vaccine is constructed by subcloning the gene of interest into a eukaryotic plasmid vector. Candidate vectors include, but are not limited to, pcDNA3, pCI, VR1012, and VR1020. This construct is used as a vaccine.

Each of the newly identified genes, ProA, ProB, ORF, the cytochrome oxidase homolog, or the partial lipoprotein signal peptidase homolog can be used to create a DNA vaccine (reviewed in Robinson, 1997). In addition, any immunologically active portion of these proteins is a potential candidate for the vaccine. A plasmid containing one of these genes in an expression vector is constructed. The gene must be inserted in the correct orientation in order for the genes to be expressed under the control of eukaryotic promoters. Possible promoters include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, the human tissue plasminogen activator (t-PA) gene (characterized in Degen et al., 1986), and the promoter/enhancer region of the human elongation factor alpha (EF-1 a) (characterized in Uetsuki et al., 1989). Orientation is identified by restriction endonuclease digestion and DNA sequencing.

Expression of these gene products is confirmed by indirect immunofluorescent staining of transiently transfected COS cells. The same plasmid without these genes is used as a controL Plasmid DNA is transformed into Escherichia coli DH5a. DNA is purified by cesium chloride gradients and the concentration is determined by a standard protocol being known in the art, and incorporated by reference (Nyika et al., 1998).

Once the vector is purified, the vector containing the DNA can be suspended in phosphate buffer saline solution and directly injected into dogs. Inoculation can be done via the muscle with a needle or intraveneously. Alternatively, a gene gun can be used to transport DNA-coated gold beads into cells by a technique known in the art, and hereby incorporated by reference (Fynan et al., 1993). The rationale behind this type of vaccine WO 01/07625 PCT/US00/19763 8 is that the inoculated host expresses the plasmid DNA in its cells, and produces a protein that raises an immune response. Each of the newly identified genes can be used to create a vaccine by this technique.

CpG molecules can be used as an adjuvant in the vaccine. This technique is known in the art, and is hereby incorporated by reference (Klinman et al., 1997).

Adjuvants are materials that help antigens or increase the immune response to an antigen.

The motifs consist of an unmethylated CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines. Oligonucleotides containing CpG motifs have been shown to activate the immune system, thereby boosting an antigen-specific immune response. This effect can be utilized in this invention by mixing the CpG oligonucleotides with the DNA vaccine, or physically linking the CpG motifs to the plasmid DNA.

Recombinant Vaccine In order to develop a recombinant vaccine, each of the genes is individually subcloned into overexpression vectors, and then purified for vaccine development. ProA, ProB, ORF, the cytochrome oxidase homolog or the partial lipoprotein signal peptidase homolog is expressed in a plasmid with a strong promoter such as the tac, T5, or T7 promoter. Alternatively, immunologically active fragments of these proteins are used in the development of a vaccine. Each of these genes is subcloned into a plasmid and transformed into an E. coli strain as described above.

The recombinant protein is overexpressed using a vector with a strong promoter.

Vectors for use in this technique include pREST (Invitrogen Inc., CA), pKK233-3 (Pharmacia, CA), and the pET system (Promega, WI), although any vector with a strong promoter can be used. After overexpression, the proteins are purified and mixed with adjuvant. Potential adjuvants include, but are not limited to, aluminum hydroxide, QuilA, or Montamide. The purified protein is used as immunogen to vaccinate dogs by a technique being known in the art, and incorporated by reference (Chang et al., 1993c; Chang et al., 1995). Briefly, the individual protein is expressed and purified from E. coli.

Then, the dogs are injected intramuscularly or subcutaneously with the purified recombinant vaccine and adjuvant. This injection elicits an immune response.

WO 01/07625 PCT/US00/19763 9 T Cell Epitope Vaccine Direct cell cytoxicity mediated by CD8 T lymphocytes (CTL) is the major mechanism of defense against intracellular pathogens. These effector lymphocytes eliminate infected cells by recognizing short peptides associated with MHC class I molecules on the cell surface. Exogenous antigens enter the endosomal pathway and are presented to CD4' T cells in association with class II molecules whereas endogenously synthesized antigens are presented to CD8 T cells in association with MHC class I molecules. E. canis is an intracellular pathogen that resides in monocytes and macrophages. The present invention develops novel ways of generating an E. canisspecific CTL response that would eliminate the organism from monocytes or macrophages of infected animals.

A strategy for increasing the protective response of a protein vaccine is to immunize with selective epitopes of the protein. The rationale behind this is that an epitope vaccine contains the most relevant immunogenic peptide components without the irrelevant portions. Therefore, a search is performed for the most highly antigenic portions of the newly identified proteins.

To identify T-cell epitopes from the newly discovered proteins, an initial electronic search for homologous sequences to known T-cell epitopes is performed. In addition, extensive T-cell epitope mapping is carried out. Each of the proteins, ProA, ProB, ORF, the cytochrome oxidase homolog, and the partial lipoprotein signal peptidase homolog, is tested for immunogenic peptide fragments. Mapping ofT cell epitopes by a technique known in the art is hereby incorporated by reference (Launois et al., 1994; Lee and Horwitz, 1999). Briefly, short, overlapping peptide sequences (9-20 amino acids) are synthesized over the entire length of the protein in question. These short peptide fragments are tested using healthy dogs which have been immunized with the protein of interest. Peripheral blood mononuclear cells from the dogs are tested for T cell stimulatory and IFN-y inducing properties. Those fragments which elicit the strongest response are the best candidates for a T-cell epitope vaccine.

WO 01107625 PCT/US00/19763 Once fragments are identified which will make the best epitopes, a recombinant adenylate cyclase ofBordetella bronchiseptica is constructed carrying an E. canis CD8' T cell epitope. The adenylate cyclase toxin (CyaA) of Bordetella bronchiseptica causes disease in dogs and elicits an immune response. In addition, CyaA is well suited for intracytoplasmic targeting. Its catalytic domain corresponding to the N-terminal 400 amino acid residues of the 1,706-residue-long protein, can be delivered to many eukaryotic cells, including cells of the immune system. Also, toxin internalization is independent of receptor-mediated endocytosis, suggesting that the catalytic domain can be delivered directly to the cytosol of target cells through the cytoplasmic membrane. The Pseudomonas aeruginosa exotoxin A (PE) is another toxin which could be used in this procedure to deliver peptides or proteins into cells, by a technique known in the art, and hereby incorporated by reference (Donnelly et al., 1993).

Foreign peptides (16 residues) have been inserted into various sites of the AC domain of CyaA without altering its stability or catalytic and calmodulin-binding properties. Thus, protein engineering allows the design and delivery of antigens that specifically stimulate CTLs. The induction of specific CD8 T cells can play an important role in canine ehrlichiosis control due to the intracellular persistence of E. canis in monocytes.

The adenylate cyclase (AC) toxin (cya) gene of B. bronchiseptica has been cloned.

A synthetic double-stranded oligonucleotide encoding a 9 to 20 amino acid class I T cell epitope of either ProA, ProB, ORF, the cytochrome oxidase homolog, or the partial lipoprotein signal peptidase homolog, is designed according to B. bronchiseptica codon usage. The complementary oligonucleotides are inserted in the hypervariable region of the cloned AC-coding sequence of the cya. This technique is known in the art in other systems, and is incorporated by reference (Sebo et al., 1995; Guermonprez et al., 1999).

Recombinant plasmids carrying the chimeric cya gene are sequenced to determine the copy number and orientation of the inserted epitope. A plasmid with a complete copy of the insert that specifies the T-cell epitope (CD8 in the correct orientation is chosen from the sequenced plasmids. The ability of the new chimeric protein to enter eukaryotic cells is necessary to ensure intracellular targeting of the epitopes (Fayolle et al., 1996).

WO 01/07625 PCT/US00/19763 11 A vaccine can be created in one of two ways. Recombinant chimeric protein can be purified and used to inoculate dogs. Alternatively, an attenuated B. bronchiseptica strain that carries a T-cell epitope or E. canis gene by in-frame insertion into adenylate cyclase is created by allelic-exchange. Allelic-exchange is a technique known in the art, and is hereby incorporated by reference (Cotter and Miller, 1994).

Finally, protection against E. canis infection in dogs vaccinated with the adenylase cyclase- ProA, ProB, ORF, cytochrome oxidase homolog, or lipoprotein signal peptidase homolog chimeric protein is determined. Wild type and recombinant ACs and CyAs are diluted to working concentrations in PBS and the chimeric protein is injected into dogs either intramuscularly or subcutaneously. Alternatively, the T-cell epitope is inserted into the adenylate cyclase gene of an attenuated B. bronchiseptica strain in frame, and the dogs are given the live bacteria.

Recombinant antigens are promising candidates for human and animal vaccination against various pathogens. However, a serious drawback is the poor immunogenicity of recombinant antigens as compared to native antigens. A major challenge in the development of a new recombinant vaccine is, therefore, to have a new adjuvant system that increases the immunogenicity of antigens. Cytokines are powerful immunoregulatory molecules. Cytokines which could be used as adjuvants in this invention include, but are not limited to, IL-12 (interleukin-12), GM-CSF (granulocyte-macrophage colony stimulating factor), IL- 1 (interleukin-lp) and y-IFN (gamma interferon).

These cytokines can have negative side effects including pyrogenic and/or proinflammatory symptoms in the vaccinated host. Therefore, to avoid the side effects of a whole cytokine protein, an alternate approach is to use synthetic peptide fragments with the desired immunostimulatory properties. The nonapeptide sequence VQGEESNDK of IL-I 3 protein is endowed with powerful immuno-enhancing properties, and is discussed here to illustrate the use of a cytokine to increase immunogenicity.

This nonapeptide is inserted into the ProA, ProB, ORF, the cytochrome oxidase homolog, or the partial lipoprotein signal peptidase homolog protein and its immunogenicity is compared to that of the native protein. Reportedly, the insertion of this WO 01/07625 PCT/US00/19763 12 sequence into a poorly immunogenic recombinant antigen increases the chance of a strong protective immune response after vaccination. This peptide could enhance the in vivo immune response against both T-dependent and T-independent antigens. The canine IL- S1 sequence may mimic many immunomodulatory activities of the entire molecule of IL- 11 while apparently lacking many of its undesirable proinflammatory properties. This strategy is employed to increase the immunogenicity of ProA, ProB, ORF, cytochrome oxidase, the partial lipoprotein signal peptidase homolog and other E. canis antigens.

Plasmid pYFC199 is derived from a pBR322 plasmid by the insertion of a fragment that includes the ProA, ProB, ORF, the cytochrome oxidase homolog, or the partial lipoprotein signal peptidase protein from E. canis. This plasmid contains a unique HindIII site where in-frame insertions encoding exogenous sequences can be inserted.

Two complementary oligonucleotides, AGGCTTGTTCAGGGTGAAGAAGAATCCAACGACAAAAGCTT and AAGCTTTTGTCGTTGGATTCTTCACCCTGAACTTGCCA, that encode the canine ILl1 163-171 peptide are annealed, cut with HindIl, and inserted into the pYFC199 HindII site. The recombinant plasmid carrying the chimeric IL-1 3 gene is sequenced to determine the orientation of the inserted epitope.

The efficacy of the recombinant proteins as vaccines is tested in dogs. The purified protein is injected intraperitoneally into dogs. Specific pathogen free (SPF) dogs are divided into five groups: one group is given recombinant adenylate cyclase of Bordetella bronchiseptica carrying E. canis CD8 T cell epitopes derived from ProA, ProB, ORF, cytochrome oxidase homolog, or the partial lipoprotein signal peptidase homolog, one group is given recombinant adenylate cyclase of Bordetella bronchiseptica as a control, one group is given the ProA, ProB, ORF, cytochrome oxidase homolog, or the partial lipoprotein signal peptidase homolog protein plus a canine IL-I1 163-171 insert, one group is given a T cell epitope derived from ProA, ProB, ORF, cytochrome oxidase homolog, or the partial lipoprotein signal peptidase homolog alone, and the last group is given PBS as a negative control.

WO 01/07625 PCT/US00/19763 13 All animals are vaccinated (30-40 g.g each) four times. The dogs are challenged ten days after the last vaccination with 10 7 E. canis. At day five postchallenge, approximately 1 ml blood from each dog is collected in an EDTA tube. Whether the vaccinated groups eliminate the organisms as compared to that of the control group is tested by culture and PCR Two primers derived from the genes cloned can be used to amplify the gene product from the tissues or blood samples from these dogs. The internal primer can also be designed for use as an oligonucleotide probe to hybridize the PCR gene product.

This invention provides a badly needed vaccine against the E. canis bacterium.

The vaccine can be used to protect dogs throughout the world from canine monocytic ehrlichiosis.

Accordingly, it is to be understood that the embodiments of the invention herein described are merely illustrative of the application of the principles of the invention.

Reference herein to details of the illustrated embodiments are not intended to limit the scope of the claims, which themselves recite those features regarded as essential to the invention.

Editorial Note Note: There are no pages numbered 14 to 31.

Editorial Note File No 62257/00 The following sequence listing is numbered 1 to 18 followed by page number 32.- 4 WO 01/07625 WO 0107625PCTIUSOO/19763

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SEQUENCE LISTING <110> <120> <130> <150> <151> <160> <170> <210> <211> <212> <213> Chang, Yung-Fu* EHRLICHIA CANIS GENES FOR VACCINE DEVELOPMENT crf 2322 U.S. 09/358,322 1999-07-21 11 Patentln Ver. 1 5300

DNA

Ehrlichia canis <400> 1 gatcaaataa tatctatgat caggt tatgg ttattaaagt aagtatctca atctacttga gaaaatattt aaaaaactat ctgctgacgt catataccgt ttatctattc tgcaactttc agaagaattt acat taacaa aatgaaacca atgtttagtg aggtacagta cagatttaaL tgtatttgta tgaggacac t taataaggta aatgccagta aaaactcatc acattataaa aa caga ttc t tatcactgat tattaaaagc tgct taatta agaataagaa tacgcttctg agaacaagta gcagatatac aaaccaggag tcaggaatgg gcttgttttt tcatttttta actctttcat ctgat taaaa tttcaattag atataaaagt tttgaatcaa aagtattatt acactattta taccactata atatatcaaa acaaacaac t aacaaaaat t ctgtatataa gtttcaccaa tagatccagc atgtattctt aaaataac ta agagtattca gaaataaatt atttaattac atttacctta tggattaata tagtatattt ttctaaaata gccatggaaa gattttctac tgt tacacca acaaacatta catagaaaca taagtacaaa ttaatattga aaaacactac taaaaaactt tgatataaaa atttcataac gcaataatac tgtaaagtaa ggtaacac ta ttctatccag cgcgcagaaa cataaagtag taccctcatc gatcctgaaa gaataaactt gcaaaataat aactactgct tagttttaat atactattaa ctttattaac WO 01/07625 WOOI/7625 CTIUSOO/19763 aatttcataa atataaactc ttttcatagt atgaaaaatt tcatgcacat tagcacattt gcacac ttag actacgaatt tgcagaattt aaagaaaaat cattttatta acaacaaaga taattgtaac ggaaaatacc aaacaaatat tgtatcaaat aaatac tcgg tagttacatc ttgaagctat gtataaataa aagtaaaagc gcatgcatct acaaagtaag gattaaccgg attaatattg agaagctaca aatttzcctta aggacttgca cagttttgca taaaaatact ccaaataaac tttcttaaca agataatgga ggtattatac tt ttgaacac taatataggc aataccaaaa taaggttacc gagagt tgaa caatggatat agttgctgaa tggagatgca atctaataat gactttaaca accaaatggt tagtggtaaa gataaaaaca acctaaaaac ttatttagaa acatttaact aatactagga tatccaagat gcatttatta attttctttt actaaaaata aaatttgcat tact ttaca t caacaat tag ttactcttat tattgcaagg atagccaatc atggaagtat aaagtcggtg ttaatgttta ggaaatttca caatatttat gacaaagcat agccaagcaa ggcagaccag gcctttcata gatccacaag aagaaacctt ttaaaagaca attaccaata ttcagcctgc gattataatt gggatc tcta aatggaattt tatgcatttg gtaccgctat gttaactccg cctaatggag cattcaatac aaatacacta tcaagaaagc caaaaatatt aaggcaaagg ttttttatca ttatggtaat atgctttatc acgtgattcc gaactgatga gtggaacaga atgcaagcac ctcttgcaat taataagaga aaaacatac t tagtaggatg agctacatta aagtaatcac caagtcaagt gttcagtaga aaaactacat tttacaatga acttaactga cagaagctgt cagcagaata acggactaac cagaaa tcag ctatggaaaa aatagttatg atatgcaaat tctatatgtt aggatacgc t aaacgaagga tatagactta cttgatatta gatgaaattt ctttaacatt aaatcatcgc tccagtagga aaaatttcct atctcaattt ggatattgaa acaaaaggta agaagaagaa ggaacatgaa tagtcctaat acttgcaaaa tagggtagaa aatcccagaa act taacatg tt t ggtaatt cagcgataat agaacaagaa ttitagaaagt tttcatatca taatatttac tatctttcaa agaaacatat gatctcaata gaacatcata tatgatgcct tcaaaaaaca aaatttgata ttaaataatc tttacttgtt aaagttacac gcaccagcag tactctggat aatctcatca tgtactatat tcagacagaa gtcttagaag atggaaaatg attagcaact aatgctatat caatactatg ccaccgcata ctgtttttaa atgttagcag aacaatccaa tacctttcca attcataaat gcaaagtata tatttttatg gataccatag aacaatataa tgtgttacac ttaacataaa acc taccaac ttgataagca actatgctct tagacctaga 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 WO 01/07625 W00117625PCT/USOO/19763 caatttttat cagtgattgc acagattgca gaatcacgct aatcaataat acaaatcgtt taaatatatt tgt taataga gtttgctaca tactatgcta aggattaaca tggtacaata tatagagcac tattaccaac aagcttacaa agcaataaca aataattgaa cataccttgg taagtaagta cggagaagct aaaaaattag acaac ttata aattaataaa tacaatttta gagcaattac ataatactaa tcatagcaca gttaggttta atatcattaa atattcaaca catgttaaat atattcaaag atcaaccagg atcagcgcag ctttccaaat gaagacacat gacacagtac ggcggattaa taccatagta ttcactgata attaaaaagt tcttttattt ttacacgatc atagaagaag gtaggaaaaa t taagtatac taagtagctt tat tataagc taatcacaat tttagaaaat acaatgagta cttactcaat tacttaatag tcagttaaaa agtatacgga gctgcattct aaacc ttatc ccgtcacaga cattatattc ggcaccc ata aagacgttgc caggagatgt tgccatctgg tattatatgt catatcacag gtctcaattc gcagttcact ataccacagt atggagttga tatctatgtt tagatccgag taaataaaat acaataacat aggttattgt caatcaaata ttttaacctg atcaaatttt gacaacagat tttttactta aaaagcagtt tatagctgtt ttttgaagtg actttcagcc tgtttgctgc agaaaacttt gaagaagccc tagttttact 2640 t caagaaa ta tgctcctgaa t tctaacaaa attatatata agatccaaca taataacaaa acagagagat caaagactat aatattaatg atc taacatg aacaaaatgt tgaagacact aaataacaat ttatattaat tgccaagaaa aaatggcaaa atttactaca aaaaaacatt ggataatatg ctttacagga acacacatca attatttaat atactaccaa gacaagccgc tttcaccata t ttagaaaga tgctgcctgc ttca ataga a tt ta tagct a gtttacggga aaaaatagt t cagc tatcaa aa ta ccatac gtaccacaaa catgcatcaa atagaattaa aatcatagta atatccgtct tttgcaattg aatgttagtg aaatacaatt attttatcta caaatagatg agtattctat aaccaaagtg aagt tttgct ttatattgtg ataaattatc tttatttt tt gtattggatg aatc tgcggt atggtattat gctgctataa agtgatcgtt taatagcaga 2700 caacagaaat 2760 cattaaatac 2820 ttgacaagga 2880 atttactaga 2940 cagatacgac 3000 gtgt cataat 3060 acttgttcaa 3120 gagacaagtt 3180 atgtgctatt 3240 taacagatat 3300 caaaatctag 3360 agatattgtt 3420 cttactacaa 3480 atgaattagt 3540 ctaaaaaaca 3600 taggttgtat 3660 ttagctctac 3720 aatgttaagc 3780 acctaccata 3840 actacaattc 3900 aaaataaaat 3960 gtattaatcg 4020 tcttgacaaa 4080 tta~tgaaagc 4140 tcattgcagt 4200 tccaaagatt 4260 WO 01/07625 WO 0107625PCTUSOO/19763 gcaattaaca ttccttaaca tattagacat aataatttct agatctattc agccgtttct agataaacaa agatcaagga tatagcttct ttatgtttta tgttatatta tttgccatta caatgtaagt tctatgatat Igtactataa acattactat gtaaagcgca taccatttac aatccatttg gcagcattaa cgctttggat ttattacttg tt tag caaga gttttcctac ac tcacgtta atgaatattt tgtgacatt t gcctgtattc tataaaacat ttattactat tat tgatata ttcctattga ataagtagca attcaaagtt agaagcttaa t tac ctga tc taatagcagg gtatatgctt acgagtcaag ctgcagcatt tgcaagaact tctcattcgt ttatattatc tgtcagcagt tagcagtgag acctttttat ctaggagtta acctaaaaat aaaatctata ccccccaata taaaacacaa atttaatata aatggttggc aactaaaagt cacttcttct ttgtggaaag ctccaatcca aatgtatgct taatcaacaa actcccagca.

cagccgggga taggtgttgt cagttaattt ataaaagaat aaa tcataga atgattacaa gtaatcaaaa ccaaaactaa cttgcagt tc aagc aagt ca tctgtactgc ataatgggta cttgttgttg gcaaagaaca actatagaag gctggcattg tcctc tcagc atcgtttctt gtttcatgtg ccgccaggt t cagcaccata gaggtaatct tcgagatac t ttccagcat t ttttagttgc cacaacatgc ttgcaatatc atgacaaccc cagctattgt ttataagtcc aagcaaaagt acatcatgac atcaatagat tatataagtg gttattactc gaatacaggc tcttattcta ataatgtggc acaaaaaaca ccacactgta 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 tatctctatt acacctttat ctcctatcaa atttactaca 5280 5300 <210> 2 <211> 5: <212> Dl <213> El <220> 22 hrlichia canis <221> CDS <222> <223> Protein translated from nucleotides 12 through 533 (cytochrome oxidase homolog).

<400> 2 W001/07625 WOOI/7625PCT/USOO/19763 atg aaa cca aga. ata aga aac act att tat gga tta ata gca ata ata Met 1 c ta Leu ttt Phe tca Ser gat Asp g ta Val aat Asn cca Pro a cc Thr ttt Phe 145 aaa Lys Ile c ta Leu aca Thr aga Arg gaa Glu tac Tyr tat Tyr t gt Cys 125 atg Met act Thr a aa Lys <210> 3 <211> 174 <212> PRT <213> Ehrlichia canis <400> 3 Met Lys Pro Arg Ile Arg Asn Thr Ile Tyr Gly Leu Ile Ala Ile Ile 1 5 10 p WO 01/07625 PCTIUSOO/19763 Leu Phe Ser Asp Val Asn Pro Thr Phe 145 Lys Leu Val Gly Tyr Gly Asn Leu Pro 70 Gly Glu Asp Thr Lys Tyr Tyr Pro 135 Ala Ile 150 Ser Tyr Ser Val Thr Val Ile Lys Phe Tyr Leu Ile Met Ala Lys Val Lys Thr Asp Pro 155 Phe Lys 170 Leu Thr Arg Giu Tyr Tyr Cys 125 Met Thr Lys <210> 4 <211> 1314 <212> DNA <213> Ehrlichia canis <220> <221> CDS <222> (1)..(1314) <223> Protein translated from nucleotides 939 through 2,252 (ProA).

<400> 4 atg atg aaa ttt ttt act tgt ttt ttc ata gtt. ttc tta aca ata gcc 48 Met met Lys Phe Phe Thr Cys Phe Phe Ile Val Phe Leu Thr Ile Ala 1 5 10 '0 WO 01/07625 PCT/U 500/19763 aat Asn aat Asn atg Met tac Tyr gaa Glu ttc Phe cca Pro cag Gin gtc Val 145 c ta Leu cca Pro gct Ala att Ile gct tta Ala Leu atg gaa Met Giu atg gta Met Val gga tta Gly Leu ttt cct Phe Pro gca agc Ala Ser 100 caa tat Gin Tryr 115 ttt aag Phe Lys gaa gaa Glu Glu gaa gaa Glu Glu gta gga Val Gly 180 gcc ttt Ala Phe 195 act gga Thr Gly tcc ttt aac att aaa gtt aca cat gaa aaa tta gat Ser Phe Asn Ile Lys Val Thr His cca aat cat cgc Pro Asn His Arg ggt gga act gat Gly Gly Thr Asp gaa cac tta atg Giu His Leu Met 75 aca ctt agt aat Thr Leu Ser Asn tgt act ata tac Cys Thr Ile Tyr 105 atg gat att gaa met Asp Ile Glu gca tta ata aga Ala Leu Ile Arg 140 gtt gaa agc caa Val Giu Ser Gin 155 ttt tat tac aat Phe Tyr Tyr Asn 170 att agc aac tac Ile Ser Asn Tyr 185 tat agt cct aat Tyr Ser Pro Asn caa gaa gta atc Gin Giu Val Ilie 220 Giu gca Ala gat Asp ttt Phe ata Ile tac Tyr tca Ser 125 gaa Giu gca Ala gga Gly aac Asn aa t Asn 205 aca Thr Leu Asp gca gtc Ala Vai gta gga Val Gly gga aca Gly Thr gga aat Gly Asn tta ata Leu Ile aga atg Arg Met aag gta Lys Val aac ata Asn Ile 160 ggc aga Gly Arg 175 gaa gtt Giu Vai ata tta Ile Leu gca aaa Ala Lys agt caa Ser Gin 240 caa tac tat ggg aaa ata cca tct aat aat aag aaa cct tca Tyr Tyr Gly Lys Pro Ser Asn Asn Lys Pro Ser WO 01/07625 WO 0107625PCT[USOO/19763 gtt agg gta gaa cca ccg cat aaa aca aat atg act tta aca tta aaa Val Arg Val Giu Pro Pro His Lys Thr 245 tca gta gaa Ser Val Giu 260 att acc aat Ile Thr Asn 275 ggt agt ggt Gly Ser Gly cca ata gtt Pro Ile Val gat aat tac Asp Asn Tyr 325 gaa gct gta Giu Ala Val.

340 aat gga att Asn Gly Ile 355 gca cat tta Ala His Leu tat ggc atg Tyr Gly Met att tac gat Ile Tyr Asp 405 atg gaa aat Met Glu Asn 420 atc Ile aaa Lys aaa Lys aca Thr 310 ct t Leu gaa Glu tca Ser act Thr cat His 390 ac c Thr atc Ile Asn Met 250 ttt tta Phe Leu ctt aac Leu Asn ctt tac Leu Tyr aca gat Thr Asp 315 gct ata Aia Ile 330 cat aaa His Lys tta gaa.

Leu Glu gac gga Asp Giy gga gta Gly Val.

395 gta agt Val Ser 410 Leu tat Tyr atg Met 285 gat Asp aat Asn aaa Lys ata Ile gca Ala 365 act Thr cta Leu caa Gin Thr Caa Gin 270 tta Leu ttg Leu tac Tyr aac Asn aat Asn 350 aag Lys t tc Phe tca Ser gat Asp 768 816 864 91i2 960 1008 1056 1104 1152 1200 1248 1296 1314 caa aac aat ata aga tta acc Gin Asn Asn Ile Arg Leu Thr ggg cat Gly His tta tta ci Leu Leu P: 4: <210> <211> 438 aat gga gaa Asn Gly Giu WO 01/07625 WO~l/7625PCTUSOO/19763 <212> PRT <213> Ehrlichia canis <400> Met 1s Asn Met Tyr Giu Phe Pro Gin Val1 145 Leu Pro Ala Ile Gin 225 Val Phe Ile Val Lys Val Thr 25 Pro Asn His Giy Giy Thr Giu His Leu Thr Leu Ser Cys Thr Ile 105 Met Asp Ile Ala Leu Ile Val Giu Ser 155 Phe Tyr Tyr 170 Ile Ser Asn 185 Tyr Ser Pro Gin Glu Val ASn Asn Lys 235 Thr Asn Met 250 Leu Giu Ala Asp Phe Ile Tyr Ser 125 Giu Aia Gly Asn Asn 205 Thr Pro Leu Thr Lys Pro Pro Ser Giy Giu 110 Asp Gin Lys Tyr Lys 190 Ala Leu Ser Thr Asp Ser Ser Asp Se Ser u Ile Pro Glu PhLeMtTr Phe Leu Met Tyr Gin Ile Pro 270 WO 01/07625 WOO1/7625PCT/USOO/19763 Asn Gly Ile Thr Asn Lys Asn Tyr Ile Leu Asn Met Met Leu Ala Glu 275 280 285 Ile Leu Gly Ser Gly Lys Phe Ser Leu Leu Tyr Asn. Asp Leu Val Ile 290 295 300 Asn Asn Pro Ile Val Thr Ser Ile Lys Thr Asp Tyr Asn Tyr Leu Thr 305 310 315 320 Asp Ser Asp Asn Tyr Leu Ser Ile Glu Ala Ile Pro Lys Asn Gly Ile 325 330 335 Ser Thr Giu Ala Val Glu Gin Glu Ile His Lys Cys Ile Asn Asn Tyr 340 345 350 Leu Glu Asn Gly Ile Ser Ala Giu Tyr Leu Giu Ser Ala Lys Tyr Lys 355 360 365 Val Lys Ala His Leu Thr Tyr Ala Phe Asp Gly Leu. Thr Phe Ile Ser 370 375 380 Tyr Phe Tyr Gly Met His Leu Ile Leu Gly Val Pro Leu Ser Giu Ile 385 390 395 400 Ser Asn Ile Tyr Asp Thr Ile Asp Lys Val Ser Ilie Gin Asp Vai Asn 405 410 415 Ser Ala Met Giu Asn Ile Phe Gin Asn Asn Ile Arg Leu Thr Gly His 420 425 430 Leu Leu Pro Asn Gly Glu 435 <210> 6 <211> 1407 <212> DNA <213> Ehriichia canis <220> <221> CDS <222> (1407) <223> Protein translated from 2,258 through 3,664 (ProB).

<400> 6 atg aga aac ata ttg tgt tac aca tta ata ttg att ttc ttt tca ttc 48 Met Arg Asn Ile Leu Cys Tyr Thr Leu Ile Leu Ilie Phe Phe Ser Phe 1. 5 10 WO 01/07625 WO 0107625PCTIUSOO/19763 aat Asn aaa Lys att Ile ttt Phe gga Gly aaa Lys tca Ser agt Ser ata Ile 145 gaa Glu cca Pro aac Asn caa Gin tat Tyr aaa Lys tta Leu aag Lys aaa Lys at a Ile aaa Lys 115 tgC Cys gca Ala ata Ile tct Ser gaa Giu 195 gtt Val aat Asn tat Tyr 40 aag Lys tac Tyr ctc Leu gat Asp aac Asn 120 gtc Val cat His atg Met ggg Gly tat Tyr 200 gga.

Gly aac Asn gaa Giu gca Ala aca Thr ttt Phe gac Asp gaa Glu gat Asp aaa Lys cac *His 170 tta Leu Laaa *Lys gta Val gaa Glu aac Asn gct Ala ata Ile c aa Gin aat Asn c ta Leu 125 ata Ile tat Tyr ttc Phe atc Ile ttt Phe 205 aca Thr gc t Al a cta Leu tat Tyr tta Leu tta Leu ttt Phe 110 gtt Val ttC Phe tct Ser aaa Lys aat Asn 190 gac Asp cag Gln aat tta cta gat aaa tat att Ctt tcc aaa ttg cca tct Asn Leu Leu Asp Lys Tyr Ile Leu Ser Lys Leu Pro Ser 225 230 235 ggt aat aac Gly Asn Asn 17 WO 01/07625 WO~l/7625PCr[USOOI19763 aaa aat acc ata cca gat acg act gtt aat aga gaa gac aca tta tta Lys Asn Thr Ile Pro Asp Thr Thr Val Asn Arg Glu Asp Thr Leu Leu gta Vai gta Val1 atg Met 290 gac Asp aa t Asn gta Val aag Lys acc Thr 370 ata Ile aaa Lys att Ile aaa Lys cct Pro 450 atg Met tca Ser tta Leu 300 agt Ser ttc Phe att Ile att Ile aac: Asn 380 gat Asp ata Ile gta Val gat Asp act Thr 460 768 816 864 912 960 1008 1056 1104 1152 1200 1248 1296 1344 1392 WO 01/07625 tta ggt tgt att aag Leu Gly Cys Ile Lys 465 <210> 7 <211> 469 <212> PRT <213> Ehriichia canis <400> 7 Met Arg Asn Ile Leu Cys 1 5 Asn Thr Tyr Ala Asn Asp Lys Asn Lys Ile His Tyr Ilie Ser Leu Lys Phe Ala Phe Asp Lys Gin Gly Leu Gly Ser Lys Asn Asn Tyr Lys Gly Ile Asp Leu Lys 100 Ser Leu Lys Thr Leu Ser 115 Ser Asp Cys Ile Phe Asn 130 Ile Ile Ala Glu Gin Ile 145 150 Giu Phe Ile Ala Thr Thr 165 Pro Tyr Ser Asn Lys Val 180 Asn Gin Glu Asp Val Ala 195 Gin Ile Val Ile Ser Aia 210 PCTIUSOO/19763 1407 Ile 10 Asn Glu Ala Thr Phe 90 Asp Giu Asp Lys His 170 Leu Lys Phe Giu Asn Ala Ile Gin Asn Leu 125 Ile Tyr Phe Ile Phe 205 Phe Ser Aia Thr Leu Pro Tyr Asp Leu Asn Leu Giu Phe Tyr 110 Vai Leu Phe Asn Ser Ala Lys Gly 175 Asn Asfl 190 Asp Lys Ala Gly Asp Val Asp 215 Thr Gin Leu Ser WO 01/07625 PCTIIJSOOI19763 14 Asn Leu Leu Asp Lys Tyr Ile Leu Ser Lys Leu Pro Ser Gly Asn Asn 225 230 235 240 Lys Asn Thr Ile Pro Asp Thr Thr Val Asn Arg Glu Asp Thr Leu Leu 245 250 255 Tyr Val Gin Arg Asp Val Pro Gin Ser Val Ile Met Phe Ala Thr Asp 260 265 270 Thr Val Pro Tyr His Ser Lys Asp Tyr His Ala Ser Asn Leu Phe Asn 275 280 285 Thr Met Leu Gly Gly Leu Ser Leu Asn Ser Ile Leu Met Ile Glu Leu 290 295 300 Arg Asp Lys Leu Giy Leu Thr Tyr His Ser Ser Ser Ser Leu Ser Asn 305 310 315 320 Met Asn His Ser Asn Val Leu Phe Giy Thr Ile Phe Thr Asp Asn Thr 325 330 335 Thr Vai Thr Lys Cys Ile Ser Vai Leu Thr Asp Ilie Ile Giu His Ile 340 345 350 Lys Lys Tyr Gly Val Asp Giu Asp Thr Phe Ala Ilie Ala Lys Ser Ser 355 360 365 Ile Thr Asn Ser Phe Ile Leu Ser Met Leu Asn Asn Asn Asn Val Ser 370 375 380 Giu Ile Leu Leu Ser Leu Gin Leu His Asp Leu Asp Pro Ser Tyr Ile 385 390 395 400 Asn Lys Tyr Asn Ser Tyr Tyr Lys Ala Ile Thr Ilie Giu Giu Val Asn 405 410 415 Lys Ile Ala Lys Lys Ile Leu Ser Asn Glu Leu Val Ile Ile Glu Vai 420 425 430 Gly Lys Asn Asn Asn Ile Asn Gly Lys Gin Ile Asp Ala Lys Lys His 435 440 445 Ile Pro Trp Leu Ser Ile Gin Val Ile Val Phe Thr Thr Ser Ile Leu 450 455 460 Leu Gly Cys Ilie Lys 465 <210> 8 <211> 675 <212> DNA <213> Ehriichia canis WO 01/07625 WO 0107625PCTIUSOO/19763 <220> <221> CDS <222> <223> Protein translated from nucleotides 4,121 through 4,795 (ORF of unknown function).

<400> 8 atg gta tta ttt atg aaa gct cat age aca agt ata. egg aac ttt cag 48 Met Val Leu Phe met Lys Ala His Ser Thr Ser Ile Arg Asn Phe Gin 1 5 10 cct tta gaa aga get get ata atc att gca gtg tta. ggt tta gct gca 96 Pro Leu Glu Arg Ala Ala Ile Ile Ile Ala Val Leu. Gly Leu Ala Ala 25 tte ttg ttt gct get gct gcc tgc agt gat cgt ttc caa aga tt~g caa 144 Phe Leu Phe Ala Ala Ala Ala Cys Ser Asp Arg Phe Gin Arg Leu Gin 40 tta aca aat eca ttt gta ata gca gga atg gtt ggc ctt gca gtt ctt 192 Leu Thx Asn Pro Phe Val Ile Ala Gly Met Val Gly Leu Ala Val Leu 55 tta gtt gct tcc tta aca gca gea tta agt ata tgc tta act aaa agt 240 Leu Val Ala Ser Leu Thr Ala Ala Leu Ser Ile Cys Leu Thr Lys Ser 70 75 aag caa gtc aca caa cat get att aga cat cgc ttt gga tac gag tea 288 Lys Gin Val Thr Gin His Ala Ile Arg His Arg Phe Gly Tyr Giu Ser 90 age act tet, tet tet gta etg ett gea ata tca ata att tet tta tta 336 Ser Thr Ser Ser Ser Val Leu Leu Ala Ile Ser Ilie Ile Ser Leu Leu 100 105 110 ctt get gca gca ttt tgt gga aag ata atg ggt aat gac aae cca gat 384 Leu Ala Ala Ala Phe Cys Gly Lys Ile Met Gly Asn Asp Asn Pro Asp 115 120 125 cta ttc ttt agc aag atg eaa gaa cte tce aat ca ctt gtt gtt gCa 432 Leu Phe Phe Ser Lys Met Gin Glu Leu Ser Asn Pro Leu Val Val Ala 130 135 140 get att gta gee gtt tet gtt tte eta cte tca tte gta atg tat get 480 Ala Ile Val Ala Val Ser Val Phe Leu Leu Ser Phe Val Met Tyr Ala 145 150 155 160 gca aag aae att ata agt cea gat aaa caa act cac gtt att ata tta 528 Ala Lys Asn Ile Ile Ser Pro Asp Lys Gin Thr His Val Ile Ile Leu 165 170 175 WO 01/07625 WO 0107625PCT/USOO/I 9763 16 tct aat caa caa act ata gaa gaa gca aaa gta gat caa gga atg aat 576 Ser Asn Gin Gin Thr Ile Giu Giu Ala Lys Val Asp Gin Gly Met Asn 180 185 190 att ttg tca gca gta ctc cca gca gct ggc att gac atc atg act ata 624 Ile Leu Ser Ala Val Leu Pro Ala Ala Gly Ile Asp Ile Met Thr Ile 195 200 205 gct tct tgt gac att tta gca gtg agc agc cgg gga tcc tct cag cat 672 Ala Ser Cys Asp Ile Leu Ala Val Ser Ser Arg Gly Ser Ser Gin His 210 215 220 caa 675 Gin 225 <210> 9 <211> 225 <212> PRT <213> Ehrlichia canis <400> 9 Met Val Leu Phe Met Lys Ala His Ser Thr Ser Ilie Arg Asn Phe Gin 1 5 10 Pro Leu Giu Arg Ala Ala Ile Ile Ile Ala Val Leu Gly Leu Ala Ala 25 Phe Leu Phe Ala Ala Ala Ala Cys Ser Asp Arg Phe Gin Arg Leu Gin 40 Leu Thr Asn Pro Phe Val Ile Ala Gly Met Val Gly Leu Ala Val Leu 55 Leu Val Ala Ser Leu Thr Ala Ala Leu Ser Ile Cys Leu Thr Lys Ser 70 75 Lys Gin Val Thr Gin His Ala Ile Arg His Arg Phe Gly Tyr Glu Ser 90 Ser Thr Ser Ser Ser Val Leu Leu Ala Ile Ser Ile Ile Ser Leu Leu 100 105 110 Leu Ala Ala Ala Phe Cys Gly Lys Ile met Gly Asn Asp Asn Pro Asp 115 120 125 Leu Phe Phe Ser Lys Met Gin Giu Leu Ser Asn Pro Leu Val Val Ala 130 135 140 Ala Ile Val Ala Val Ser Val Phe Leu Leu Ser Phe Vai Met Tyr Ala 145 150 155 160 WO1/07625 PCT/U 17 Ala Lys Asn Ile Ile Ser Pro Asp Lys Gin Thr His Val Ile Ile Leu 165 170 175 Ser Asn Gin Gin Thr Ile Glu GlU Ala LYS Val1 Asp Gin Gly Met Asn 180 185 190 Ile Leu Ser Ala Val Leu Pro Ala Ala Gly Ile Asp Ile Met Thr Ile 195 200 205 Ala Ser CyS Asp Ile Leu Ala Val Ser Ser Arg Gly Ser Ser Gin His 210 215 220 Gin 225 <210> <211> 417 <212> DNA <213> Ehrlichia canis <220> <221> CDS <222> <223> Protein translated from complementary sequence derived from nucleotides 4,884 to 5,300 (partial lipoprotein signal peptidase homolog).

<400> gat cag gta agt aaa tgg tat gta gta aat ttg ata. gga gat aaa. ggt Asp Gin Val Ser Lys Trp Tyr Val Val Asn Leu Ile Gly Asp Lys Gly 1 5 10 gta ata gag ata tta ago ttc ttg cgc ttt act aca gtg tgg aat gct Val Ile Glu Ile Leu Ser Phe Leu Arg Phe Thr Thr Val Trp Asn Ala 25 gga att agt ttt ggt ata tta aat aac ttt gaa tat agt aat gtt gtt Gly Ile Ser Phe Gly Ile Leu Asn Asn Phe Glu Tyr Ser Asn Val Val 40 ttt tgt agt atc tcg att ttg att act tgt gtt tta tgc tac tta ttt Phe Cys Ser Ile Ser Ile Leu Ile Thr Cys Val Leu Cys Tyr Leu Phe 55 ata gta cag cca cat tat aga tta cct ctt gta atc att att ggg ggg Ile Val Gin Pro His Tyr Arg Leu Pro Leu Val Ilie Ile Ile Gly Gly 70 75 SOO/19763 WO 01/07625 WO 0107625PCTUJSOO/19763 gga aat atc ata Gly Asn Ile Ilie gat ttt tat atc Asp Phe Tyr Ile 100 tct ttt ata ttt Ser Phe Ile Phe 115 cac atg aaa caa His Met Lys Gin gat aga ata Asp Arg Ile aat aac tta Asn Asn Leu 105 tta ggt ata.

Leu Giy Ile 120 att aac Lgt Ile Asn Cys 135 aga tat, ggt gct gtc tat gat Arg Tyr Gly Ala Val Tyr ASP 90 cat tgg cct gta ttc aac ctg His Trp Pro Val Phe Asn Leu 110 gta ata ata atg gca aag agt Val Ile Ile Met Ala Lys Ser 125 aac tcc Asn Ser <210> 11 <211> 139 <212> PRT <213> Ehriichia canis <400> 11 Asp Gin Val Ser Lys T: 1 5 Val Ile Glu Ile Leu S4 Gly Ile Ser Phe Gly 1' Phe Cys Ser Ile Ser 1I Ile Val Gin Pro His T4 Ser Ile Gly Asn Ile I' Phe Ile ASP Phe Tyr I' 100 Ala Asp Ser Phe Ile PJ 115 Asn Asn His Met Lys G' 130 Asn Leu Phe Thr Phe Giu Cys Val Leu Val Arg Tyr His Trp Val Ile Asn Ser

Claims (33)

1. A recombinant DNA comprising said DNA selected from the group consisting of: a) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 3; b) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. c) a recombinant DNA that encodes a protein having an amino acid sequence as shown in amino acids identified as 1 through 449 of SEQ. ID. NO. 7; d) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 9; e) a recombinant DNA that encodes a protein having an amino acid sequence as shown in amino acids identified as 1 through 31 of SEQ. ID. NO. 11; and f) a recombinant DNA that encodes a protein having an amino acid sequence as shown in S* amino acids identified as 33 through 139 ofSEQ. ID. NO. 11. •15 2. The recombinant DNA of claim 1 wherein said DNA encodes at least one immunogenic epitope.

3. A recombinant protein comprising said protein selected from the group consisting of: a) a protein having an amino acid sequence as shown in SEQ. ID. NO. 3; b) a protein having an amino acid sequence as shown in SEQ. ID. NO. S c) a protein having an amino acid sequence as shown in amino acids identified as 1 20 through 449 of SEQ. ID. NO. 7; d) a protein having an amino acid sequence as shown in SEQ. ID. NO. 9; e) a protein having an amino acid sequence as shown in amino acids identified as 1 through 31 of SEQ. ID. NO. 11; and f) a recombinant DNA that encodes a protein having an amino acid sequence as shown in amino acids identified as 33 through 139 ofSEQ. ID. NO. 11.

4. The recombinant protein of claim 3 wherein said protein includes at least one immunogenic epitope. A vaccine for protecting dogs against E. canis comprising: a) a vector capable of expressing a recombinant DNA inserted into said vector such that a recombinant protein is expressed when said vector is provided in an appropriate host; and b) the recombinant DNA inserted into said vector wherein said DNA is selected from the group consisting of: i. a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 3; 15 ii. a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. iii. a recombinant DNA that encodes a protein having an amino acid sequence as shown in amino acids identified as 1 through 449 of SEQ. ID. NO. 7; iv. a recombinant DNA that encodes a protein having an amino acid sequence as 20 shown in SEQ. ID. NO. 9; o a recombinant DNA that encodes a protein having an amino acid sequence as ••shown in amino acids identified as 1 through 31 of SEQ. ID. NO. 11; vi. a recombinant DNA that encodes a protein having an amino acid sequence as shown in amino acids identified as 33 through 139 of SEQ. ID. NO. 11; and vii. a portion of any of said recombinant DNA above that encodes a polypeptide of at least twelve sequential amino acids from said amino acids sequences above, wherein said polypeptide is sufficient to stimulate an immune response.

6. The vaccine of claim 5, wherein said DNA further comprises DNA that encodes CpG motifs.

7.The vaccine of claim 5 wherein said DNA further comprises a promoter selected from the group consisting of: a) a cytomegalovirus (CMV) immediate early promoter; b) a human tissue plasminogen activator gene and c) a promoter/enhancer region of a human elongation factor alpha (EF-1 a

8.The vaccine of claim 5, wherein said vector is selected from the group consisting of: S.p a) pcDNA3; b) pC1; 15 c) VR1012;and d) VR1020.

9.The vaccine of claim 5 wherein said vaccine is administered into said host by a method selected 0 0: from the group consisting of: a) intramuscular injection; 20 b) intraveneous injection; and c) gene gun injection. vaccine of claim 9, wherein said host is a dog.

11. A vaccine for protection against E. canis comprising: a) a recombinant protein that is selected from the group consisting of: i. a protein having an amino acid sequence as shown in SEQ. ID. NO. 3; ii. a protein having an amino acid sequence as shown in SEQ. ID. NO. iii. a protein having an amino acid sequence as shown in amino acids identified as 1 through 449 of SEQ. ID. NO. 7; iv. a protein having an amino acid sequence as shown in SEQ. ID. NO. 9; v. a protein having an amino acid sequence as shown in amino acids identified as 1 through 31 of SEQ. ID. NO. 11; vi. a protein having an amino acid sequence as shown in amino acids identified as 33 through 39 of SEQ. ID. NO. 11; and vii. a portion of any of said recombinant DNA above that encodes a polypeptide of at least twelve sequential amino acids from said amino acids sequences above, wherein said polypeptide is sufficient to stimulate an immune response.

12.The vaccine of 11, wherein said vaccine further comprises adjuvants selected from the group consisting of: a) aluminum hydroxide; b) QuilA; and 20 c) Montamide.

13. The vaccine of claim 11 further comprising a cytokine operatively associated with said recombinant protein.

14. The vaccine of claim 13 wherein said cytokine is selected from the group consisting of: a) interleukin-lp (IL-13); b) granulocyte-macrophage colony stimulating factor (GM-CSF); c) gamma interferon (y-IFN); d) amino acids VQGEESNDK from the IL-I3 protein; and e) any portion of any of the cytokines above that elicits an improved immunogenic response against E. canis. vaccine of claim 11 wherein said vaccine is administered into a host by a method selected from the group consisting of: a) intramuscular injection; and b) subcutaneous injection.

16.The vaccine of claim 15 wherein said host is a dog. S17 A vaccine for protecting dogs from E.canis comprising a recombinant protein that includes a T cell epitope wherein said T cell epitope comprises an amino acid peptide fragment of a protein selected from the group consisting of: a) a protein having an amino acid sequence as shown in SEQ. ID. NO. 3; b) a protein having an amino acid sequence as shown in SEQ. ID. NO. c) a protein having an amino acid sequence as shown in amino acids identified as 1 through 449 of SEQ. ID. NO. 7; d) a protein having an amino acid sequence as shown in SEQ. ID. NO. 9; d) a protein having an amino acid sequence as shown in SEQ. ID. NO. 9; e) a protein having an amino acid sequence as shown in amino acids identified as 1 through 31 of SEQ. ID. NO. 11; f) a protein having an amino acid sequence as shown in amino acids identified as 33 through 139 of SEQ. ID. NO. 11; and g) a portion of any of said recombinant DNA above that encodes a polypeptide of at least twelve sequential amino acids from said amino acids sequences above, wherein said polypeptide is sufficient to stimulate an immune response.

18. The vaccine of claim 17 wherein said amino acid peptide fragment comprises nine to twenty amino acids.

19. The vaccine of claim 17 further comprising a recombinant DNA encoding a protein which is capable of being internalized into eukaryotic cells, including cells of the immune system. The vaccine of claim 19 wherein said protein capable of being internalized into eukaryotic cells comprises a toxin selected from the group consisting of: a) a recombinant adenylate cyclase of Bordetella bronchiseptica; and 15 b) a recombinant exotoxin A (PE) of Pseudomonas aeruginosa.

21. The vaccine of claim 17 wherein said vaccine is administered into a host by a method selected from the group consisting of: intramuscular injection; and b) subcutaneous injection.

22. The vaccine of claim21 wherein said host is a dog. S 23. A method of identifying a T cell epitope against E. canis comprising: a) synthesizing overlapping peptide fragments over an entire length of a protein wherein said protein is selected from the group consisting of: i. a protein having an amino acid sequence as shown in SEQ. ID. NO. 3; ii. a protein having an amino acid sequence as shown in SEQ. ID. NO. iii. a protein having an amino acid sequence as shown in amino acids identified as 1 through 449 of SEQ. ID. NO. 7; iv. a protein having an amino acid sequence as shown in SEQ. ID. NO. 9; v. a protein having an amino acid sequence as shown in amino acids identified as 1 through 31 of SEQ. ID. NO. 11; vi. a protein having an amino acid sequence as shown in amino acids identified as 33 through 139 of SEQ .ID. NO. 11; and vii. a portion of any of said recombinant DNA above that encodes a polypeptide of at least twelve sequential amino acids from said amino acids sequences above, wherein said polypeptide is sufficient to stimulate an immune response; S.. S 15 b) testing said peptide fragment to determine if said peptide fragment elicits an immune response in a host animal; and c) identifying said peptide fragment as said T cell epitope of E. canis if said fragment elicits an immune response.

24.The method of claim 23 wherein said peptide fragment comprises nine to twenty amino acids. ••go

25. A method of creating a vaccine against E. canis comprising: a) selecting a vector capable of expressing a recombinant DNA inserted into said vector; and b) inserting a recombinant DNA into said vector such that a recombinant protein is expressed when said vector is provided in an appropriate host wherein said DNA is selected from the group consisting of: i. a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 3; ii. a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. iii. a recombinant DNA that encodes a protein having an amino acid sequence as shown in amino acids identified as 1 through 449 of SEQ. ID. NO. 7; iv. a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 9; v. a recombinant DNA that encodes a protein having an amino acid sequence as shown in amino acids identified as 1 through 31 of SEQ. ID. NO. 11; vi. a recombinant DNA that encodes a protein having an amino acid sequence as shown in amino acids identified as 33 through 139 of SEQ. ID. NO. 11; and vii. a portion of any of said recombinant DNA above that encodes a polypeptide of at least twelve sequential amino acids from said amino acids sequences above, wherein said polypeptide is sufficient to stimulate an immune response.

26. The method of claim 25, wherein said DNA further comprises DNA that encodes CpG motifs.

27.The method of claim 25 wherein said DNA further comprises a promoter selected from the group consisting of: a) a cytomegalovirus (CMV) immediate early promoter; b) a human tissue plasminogen activator gene and *i c) a promoter/enhancer region of a human elongation factor alpha (EF-1 a).

28.The method of claim 25, wherein said vector is selected from the group consisting of: a) pcDNA3; b) pC1; c) VR1012; and d) VR1020.

29. The method of claim 25 wherein said vaccine is injected into said host in a manner selected from the group consisting of: a) intramuscular injection; b) intraveneous injection; and c) gene gun injection. method of claim 29, wherein said host is a dog. 31 .A method of creating a vaccine against E. canis comprising: a) selecting a vector capable of expressing a recombinant protein inserted into said vector; 15 b) insertion of a recombinant DNA into said vector such that said recombinant protein is expressed when said vector is transformed into a bacterial strain wherein said DNA is selected from the group consisting of: i. a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 3; *i ii. a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. iii. a recombinant DNA that encodes a protein having an amino acid sequence as shown in amino acids identified as 1 through 449 of SEQ. ID. NO. 7; iv. a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 9; v. a recombinant DNA that encodes a protein having an amino acid sequence as shown in amino acids 1 through 31 of SEQ. ID. NO. 11; vi. a recombinant DNA that encodes a protein having an amino acid sequence as shown in amino acids 33 through 139 of SEQ. ID. NO. 11; and vii. a portion of any of said recombinant DNA above that encodes a polypeptide of at least twelve sequential amino acids from said amino acids sequences above, wherein said polypeptide is sufficient to stimulate an immune response; and c) harvesting said recombinant protein from said bacterial strain.

32.The method of claim 31, wherein said vaccine further comprises adjuvants selected from the group consisting of: ii a) aluminum hydroxide; b) QuilA; and c) Montamide.

33.The method of claim 31, wherein said vaccine further comprises a promoter selected from the group consisting of: Sa) tac; 42 b) T5; and c) T7.

34. The method of claim 31, wherein said bacterial strain is E. coli. method of claim 31, wherein said vector is selected from the group consisting of: a) pREST; b) pET; and c) pKK233-3.

36. The method of claim 31 wherein said vaccine further comprises a cytokine operatively associated with said vaccine.

37. The method of claim 36 wherein said cytokine is selected from the group consisting of: a) interleukin-lp (IL- 1); b) granulocyte-macrophage colony stimulating factor (GM-CSF); c) gamma interferon (y-IFN); d) amino acids VQGEESNDK from the IL-Ip protein; and *o• 15 e) any portion of any of the cytokines above that elicits an improved immunogenic response against E. canis.

38. The method of claim 31 wherein said vaccine is injected into said host in a manner selected from the group consisting of: a) intramuscular injection; and b) subcutaneous injection. •oooo

39. The method of claim 38 wherein said host is a dog. A method of creating a T cell epitope vaccine comprising: a) selecting a recombinant protein that includes a T cell epitope wherein said T cell epitope comprises an amino acid peptide fragment of a protein selected from the group consisting of: i. a protein having an amino acid sequence as shown in SEQ. ID. NO. 3; ii. a protein having an amino acid sequence as shown in SEQ. ID. NO. iii. a protein having an amino acid sequence as shown in amino acids identified as through 449 of SEQ. ID. NO. 7; iv. a protein having an amino acid sequence as shown in SEQ. ID. NO. 9; v. a protein having an amino acid sequence as shown in amino acids identified as 1 through 31 of SEQ. ID. NO. 11; vi. a protein having an amino acid sequence as shown in amino acids identified as 33 through 139 of SEQ. ID. NO. 11; and i:.i 15 vii. a portion of any of said recombinant DNA above that encodes a polypeptide of at least twelve sequential amino acids from said amino acids sequences above, wherein said polypeptide is sufficient to stimulate an immune response; b) identifying said T cell epitope from said protein; c) incorporating said T cell epitope into a construct capable of expressing said epitope as a 20 protein; and d) harvesting said protein. 44

41. The method of claim 40 wherein said amino acid peptide fragment comprises nine to twenty amino acids.

42. The method of claim 40 wherein said construct capable of expressing said epitope further comprises a recombinant DNA encoding a protein which is capable of being internalized into eukaryotic cells, including cells of the immune system.

43. The method of claim 42 wherein said protein capable of being internalized into eukaryotic cells comprises a toxin selected from the group consisting of: a) a recombinant adenylate cyclase of Bordetella bronchiseptica; and b) a recombinant exotoxin A (PE) of Pseudomonas aeruginosa.

44. The method of claim 40 wherein said vaccine is injected into said host in a manner selected from the group consisting of: a) intramuscular injection; and b) subcutaneous injection. The method of claim 44 wherein said host is a dog. 15 Dated this eighteenth day of March 2003 Cornell Research Foundation, Inc. By their Patent Attorneys CULLEN CO o* o