WO2000031270A1

WO2000031270A1 - Bartonella proteins and uses thereof

Info

Publication number: WO2000031270A1
Application number: PCT/US1999/027344
Authority: WO
Inventors: Kevin L. Karem; Svena Mcgill; Russell L. Regnery
Original assignee: The Government Of The United States Of America Represented By The Secretary Of The Department Of Health And Human Services
Priority date: 1998-11-19
Filing date: 1999-11-18
Publication date: 2000-06-02
Also published as: AU1734800A

Abstract

The present invention relates to Bartonella Bh83 proteins; to Bartonella Bh83 nucleic acid molecules, including those that encode such Bartonella Bh83 proteins; to antibodies raised against such Bartonella Bh83 proteins; and to uses of such proteins, nucleic acid molecules and antibodies.

Description

BARTONELLA PROTEINS AND USES THEREOF

Field of the Invention

The present invention relates to Bartonella proteins, nucleic acid molecules encoding such proteins, and antibodies raised against such proteins. The present invention also includes therapeutic and diagnostic compositions comprising such nucleic acid molecules, proteins, and antibodies.

Background of the Invention

Cat scratch disease (CSD) has been the subject of considerable clinical and microbiological interest for many years. An estimated 24,000 cases of CSD occur each year in the United States, and CSD is responsible for up to 2,000 human hospitalizations. CSD is described as a subacute regional lymphadenitis temporally associated with the scratch or bite of a cat, which occasionally results in meningoencephalitis. Very serious sequelae of CSD have been reported in immunocompromised individuals. The inventors, however, are not aware of any reports of clinical disease in cats despite the fact that cats are the reservoir for the etiologic agent of CSD.

The etiologic agent of CSD is the bacterium Bartonella henselae (B. henselae), formerly called Rochalimaea henselae. See, for example, Regnery et al., 1992, "Serological response to 'Rochalimaea henselae' antigen in suspected cat-scratch disease," The Lancet, Vol. 339, pp. 1443-1446; Regnery et al., 1992, "Naturally occurring 'Rochalimaea henselae' infection in domestic cat," The Lancet, Vol.340, pp. 557-558; U.S. Patent No. 5,399,485, by Anderson et al., issued March 21 , 1995 (Anderson et al., '485); and U.S. Patent No. 5,644,047, by Anderson etal., issued July 1 , 1997 (Anderson etal., '047). Anderson etal., '485, ibid, and Anderson etal., '047, ibid, are each incorporated herein by reference in its entirety. Treatment with antibiotics does not appear to affect the outcome of cat scratch disease. Because cats are the reservoir for B. henselae, they are the source of infection in humans. As such, exposure of humans to 6. henselae infection may best be controlled by controlling the bacterium in cats, especially pet cats that have frequent contact with humans.

As such, there remains a need to identify efficacious compositions and methods for diagnosing Bartonella infection as well as compositions and methods for protecting animals against infection by Bartonella.

Summary of the Invention

One embodiment of the present invention is an isolated Bartonella Bh83 protein. Such a protein can be a β. henselae Bh83 protein of about 83 kilodaltons (kDa) (i.e., a B. henselae Bh83 protein of 83 kDa), or a protein comprising a homologue of a β. henselae Bh83 protein of 83 kDa, wherein said homologue comprises at least one epitope that elicits an immune response against a B. henselae Bh83 protein of 83 kDa. The molecular weight is preferably determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, using a 4 to 20% gradient polyacrylamide Tris-Glycine gel.

The present invention includes Bartonella Bh83 proteins which are immunoreactive with serum from a patient having antibodies against β. henselae and/or B. quintana, but which are not immunoreactive with serum against at least one of the following organisms: Rickettsia rickettsii, Chlamydia, Treponema pallidum, Orientia tsutsugamushi, Fransciscella tularensis, Ehriichia chaffeensis, Mycoplasma pneumoniae and Escherichia coli. In one embodiment, such proteins have a molecular weight of about 83 kDa, as determined by SDS-PAGE under reducing conditions using a 4 to 20% gradient polyacrylamide Tris-Glycine gel. Other embodiments of the present invention include a nucleic acid molecule encoding a Bartonella Bh83 protein and a Bartonella Bh83 protein produced by cultu ng a recombinant cell transformed with such a nucleic acid molecule. Additional embodiments include recombinant viruses, recombinant molecules, and recombinant cells comprising such nucleic acid molecules as well as antibodies that selectively bind to a Bartonella Bh83 protein of the present invention.

Brief Description of The Drawings

FIG.1a: Western blot analysis of sera IgG (H&L) activity reacting with SDS-PAGE separated proteins of whole-cell Vero-grown B. henselae antigen preparations.

FIG. 1b: Western blot analysis of sera IgG (H&L) activity reacting with SDS-PAGE separated proteins of whole-cell agar-grown β. henselae antigen preparations.

Detailed Description of the Invention

The present invention provides for isolated Bartonella Bh83 proteins that are immunoreactive with serum from a patient having antibodies against β. henselae. A Bartonella Bh83 protein is a β. henselae protein of about 83 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions using a 4 to 20% gradient polyacrylamide Tris-Glycine gel, or any homologue thereof. A Bartonella Bh83 protein can be a full-length protein or any fragment thereof that elicits an immune response against a B. henselae Bh83 protein of 83 kDa. As such, a Bartonella Bh83 protein includes any protein from any species of Bartonella that cross- reacts with an antibody against a B. henselae Bh83 protein of 83 kDa and/or that elicits the production of an antibody that selectively binds to a β. henselae Bh83 protein of 83 kDa.

In particular, the present invention includes an isolated B. henselae protein of about 83 kDa, as determined by SDS-PAGE under reducing conditions using a 4 to 20% gradient polyacrylamide Tris-Glycine gel, and homologues of such a protein, wherein the homologue comprises at least one epitope that elicits an immune response against a β. henselae Bh83 protein of 83 kDa. The present invention also includes nucleic acid molecules that encode such proteins and antibodies directed against such proteins. As used herein, the terms isolated Bartonella proteins and isolated Bartonella nucleic acid molecules encoding such proteins refer to proteins and nucleic acid molecules derived from Bartonella', as such, the proteins and nucleic acid molecules can be isolated from an organism or prepared recombinantly or synthetically. As used herein, the term "cat scratch disease" refers to the group of diseases most normally caused by the bacterium β. henselae. Some animals (e.g., domestic cats), do not get a disease per se when infected with β. henselae and as such, "β. henselae infection" is used herein to denote infection with this bacterium, either with or without the subsequent development of detectable disease. As used herein, the term "to protect" includes, for example, to prevent or to treat (e.g., reduce or cure) B. henselae infection in the subject animal. As such, a therapeutic composition of the present invention can be a prophylactic vaccine or a treatment for animals already infected with the organism.

It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, a β. henselae protein refers to one or more B. henselae proteins, or at least one B. henselae protein. As such, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein.

An isolated Bartonella Bh83 protein of the present invention includes a β. henselae protein that is removed from its natural milieu. As such, the term "isolated" does not describe any specific level of purity of the isolated protein. As used herein, a Bartonella Bh83 protein is a β. henselae protein having a molecular weight of about 83 kDa, as determined by SDS-PAGE using a 4 to 20% gradient polyacrylamide Tris-Glycine gel, that selectively binds to serum from patients having antibodies against B. henselae (i.e., a B. henselae Bh83 protein of 83 kDa) or a homologue of such a protein. Such a protein preferably binds to IgG or IgA antibodies, more preferably to IgG_! antibodies, from patients having antibodies against β. henselae (i.e., a B. henselae Bh83 protein of 83 kDa) or a homologue of such a protein. The original identification of a β. henselae Bh83 protein of 83 kDa is detailed in the Examples. Such a protein, while immunoreactive with serum from patients having antibodies against B. henselae, is not immunoreactive with serum against an organism selected from the group consisting of: Rickettsia rickettsii, Chlamydia spp., Treponema pallidum, Orientia tsutsugamushi, Fransciscella tularensis, Ehriichia chaffeensis, Mycoplasma pneumoniae and Escherichia coli. As used herein, the terms "selectively binds to" or "is immunoreactive with" a Bartonella Bh83 protein refers to the ability of antibodies of the present invention to preferentially bind to specified proteins and mimetopes thereof of the present invention

As used herein, an isolated Bartonella Bh83 protein of the present invention can be a full-length protein or any portion (i.e., fragment) thereof that elicits an immune response against a B. henselae Bh83 protein of 83 kDa. An isolated protein of the present invention, including a homologue, can be identified in a straight-forward manner by the protein's ability to elicit an immune response against a B. henselae Bh83 protein of 83 kDa or by the protein's activity using techniques known to those of skill in the art. As used herein, a protein's ability to elicit an immune response against a β. henselae Bh83 protein of 83 kDa includes the ability to elicit a humoral and/or cellular immune response against Bartonella Bh83 protein.

As used herein, a homologue of a Bartonella Bh83 protein of the present invention can include a full-length Bartonella Bh83 protein or any portion (i.e., fragment) of a Bartonella Bh83 protein that elicits an immune response against a β. henselae Bh83 protein of 83 kDa. Examples of Bartonella Bh83 homologue proteins include Bartonella Bh83 proteins in which amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation and/or addition of glycerophosphatidyl inositol) such that the homologue includes at least one epitope capable of eliciting an immune response against a B. henselae Bh83 protein of 83 kDa, and/or of binding to an antibody directed against a β. henselae Bh83 protein of 83 kDa. For example, when the homologue is administered to an animal as an immunogen, using techniques known to those skilled in the art, the animal will produce an immune response against at least one epitope of a natural β. henselae Bh83 protein of 83 kDa. The ability of a protein to effect an immune response can be measured using techniques known to those skilled in the art. As used herein, the term "epitope" refers to the smallest portion of a protein or other antigen capable of selectively binding to the antigen binding site of an antibody or a T cell receptor. It is well accepted by those skilled in the art that the minimal size of a protein epitope is about four to six amino acids. As is appreciated by those skilled in the art, an epitope can include amino acids that naturally are contiguous to each other as well as amino acids that, due to the tertiary structure of the natural protein, are in sufficiently close proximity to form an epitope. According to the present invention, an epitope includes a portion of a protein comprising at least about 4 amino acids, at least about 5 amino acids, at least about 6 amino acids, at least about 10 amino acids, at least about 15 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 30 amino acids, at least about 35 amino acids, at least about 40 amino acids, or at least about 50 amino acids.

Bartonella Bh83 homologue proteins can be the result of natural allelic variation or natural mutation. Bartonella Bh83 homologue proteins of the present invention can also be produced using techniques known in the art including, but not limited to, direct modifications to the protein or modifications to the gene encoding the protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis.

A preferred Bartonella Bh83 protein of the present invention is a compound that, when administered to an animal in an effective manner, is capable of protecting that animal from Bartonella infection, and preferably from β. henselae or B. quintana infection. In one embodiment, such a protein protects an animal from cat scratch disease. In accordance with the present invention, the ability of a Bartonella Bh83 protein of the present invention to protect an animal from Bartonella infection refers to the ability of that protein to, for example, treat, ameliorate and/or prevent Bartonella infection, and preferably β. henselae or β. quintana infection. In one embodiment of the present invention, an isolated Bartonella Bh83 protein is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions to a gene encoding a Bartonella Bh83 protein. The minimal size of a Bartonella Bh83 protein of the present invention is a size sufficient to be encoded by a nucleic acid molecule capable of forming a stable hybrid (i.e., hybridizing under stringent hybridization conditions) with the complementary sequence of a nucleic acid molecule encoding the corresponding natural protein. The size of a nucleic acid molecule encoding such a protein is dependent on the nucleic acid composition and the percent homology between the Bartonella Bh83 nucleic acid molecule and the complementary nucleic acid sequence. It can easily be understood that the extent of homology required to form a stable hybrid under stringent conditions can vary depending on whether the homologous sequences are interspersed throughout a given nucleic acid molecule or are clustered (i.e., localized) in distinct regions on a given nucleic acid molecule.

The minimal size of a nucleic acid molecule capable of forming a stable hybrid with a gene encoding a Bartonella Bh83 protein is typically at least about 12 to about 15 nucleotides in length if the nucleic acid molecule is GC-rich and at least about 15 to about 17 bases in length if it is AT-rich. The minimal size of a nucleic acid molecule used to encode a Bartonella Bh83 protein homologue of the present invention is from about 12 to about 18 nucleotides in length. Thus, the minimal size of a Bartonella Bh83 protein homologue of the present invention is from about 4 to about 6 amino acids in length. There is no limit, other than a practical limit, on the maximal size of a nucleic acid molecule encoding a Bartonella Bh83 protein of the present invention because a nucleic acid molecule of the present invention can include a portion of a gene, an entire gene, or multiple genes. The preferred size of a protein encoded by a nucleic acid molecule of the present invention depends on whether a full-length, fusion, multivalent, or functional portion of such a protein is desired. Stringent hybridization conditions are determined based on defined physical properties of the gene to which the nucleic acid molecule is being hybridized, and can be defined mathematically. Stringent hybridization conditions are those experimental parameters that allow an individual skilled in the art to identify significant similarities between heterologous nucleic acid molecules. These conditions are well known to those skilled in the art. See, for example, Sambrook, etal., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press; Ausubel et al. /'Current Protocols in Molecular Biology," John Wiley and Sons, New York 1987 (updated quarterly); and Meinkoth, et al., 1984, Anal. Biochem. 138, 267-284, each of which is incorporated by reference herein in its entirety. As explained in detail in the cited references, the determination of hybridization conditions involves the manipulation of a set of variables including the ionic strength (M, in moles/liter), the hybridization temperature (°C), the concentration of nucleic acid helix destabilizing agents (such as formamide), the average length of the shortest hybrid duplex (n), and the percent G + C composition of the fragment to which an unknown nucleic acid molecule is being hybridized. For nucleic acid molecules of at least about 150 nucleotides, these variables are inserted into a standard mathematical formula to calculate the melting temperature, or T_m, of a given nucleic acid molecule. As defined in the formula below, T_m is the temperature at which two complementary nucleic acid molecule strands will disassociate, assuming 100% complementarity between the two strands:

T_m=81.5°C + 16.6 log M + 0.41 (%G + C) - 500/n - 0.61 (%formamide).

For nucleic acid molecules smaller than about 50 nucleotides, hybrid stability is defined by the dissociation temperature (T_d), which is defined as the temperature at which 50% of the duplexes dissociate. For these smaller molecules, the stability at a standard ionic strength is defined by the following equation:

T_d = 4(G + C) + 2(A + T). A temperature of 5°C below T_d is used to detect hybridization between perfectly matched molecules

Also well known to those skilled in the art is how base-pair mismatch (i e , differences between two nucleic acid molecules being compared, including non- complementarity of bases at a given location, and gaps due to insertion or deletion of one or more bases at a given location on either of the nucleic acid molecules being compared) will affect T_m or T_d for nucleic acid molecules of different sizes For example, T_m decreases about 1°C for each 1 % of mismatched base-pairs for hybrids greater than about 150 bp, and T_d decreases about 5°C for each mismatched base-pair for hybrids below about 50 bp Conditions for hybrids between about 50 and about 150 base-pairs can be determined empirically and without undue experimentation using standard laboratory procedures well known to those skilled in the art These simple procedures allow one skilled in the art to set the hybridization conditions (by altering, forexample, the salt concentration, the formamide concentration, orthe temperature) so that only nucleic acid hybrids with greater than a specified % base-pair mismatch will hybridize Stringent hybridization conditions are commonly understood by those skilled in the art to be those experimental conditions that will allow about 30% base-pair mismatch (i e , about 70% identity) Because one skilled in the art can easily determine whether a given nucleic acid molecule to be tested is less than or greater than about 50 nucleotides, and can therefore choose the appropriate formula for determining hybridization conditions, he or she can determine whether the nucleic acid molecule will hybridize with a given gene under stringent hybridization conditions and similarly whether the nucleic acid molecule will hybridize under conditions designed to allow a desired amount of base pair mismatch

Hybridization reactions are often carried out by attaching the nucleic acid molecule to be hybridized to a solid support such as a membrane, and then hybridizing with a labeled nucleic acid molecule, typically referred to as a probe, suspended in a hybridization solution Examples of common hybridization reaction techniques include, but are not limited to, the well-known Southern and northern blotting procedures. Typically, the actual hybridization reaction is done under non-stringent conditions (i.e., at a lower temperature and/or a higher salt concentration) and then high stringency is achieved by washing the membrane in a solution with a higher temperature and/or lower salt concentration in order to achieve the desired stringency.

For example, if the skilled artisan wished to identify a nucleic acid molecule that hybridizes under stringent hybridization conditions with a Bartonella Bh83 nucleic acid molecule of about 150 bp in length, the following conditions could preferably be used. The average G + C content of Bartonella DNA is about 40%. The unknown nucleic acid molecules would be attached to a support membrane, and the 150 bp probe would be labeled, e.g. with a radioactive tag. The hybridization reaction could be carried out in a solution comprising 2X SSC and 0% formamide, at a temperature of about 37°C (low stringency conditions). Solutions of differing concentrations of SSC can be made by one of skill in the art by diluting a stock solution of 20X SSC (175.3 gram NaCl and about 88.2 gram sodium citrate in 1 liter of water, pH 7) to obtain the desired concentration of SSC. In order to achieve high stringency hybridization, the skilled artisan would calculate the washing conditions required to allow up to 30% base-pair mismatch. For example, in a wash solution comprising 1 X SSC and 0% formamide, the T_m of perfect hybrids would be about 81°C:

81.5°C + 16.6 log (.15M) + (0.41 x 40) - (500/150) - (0.61 x O) = 81°C.

Thus, to achieve hybridization with nucleic acid molecules having about 30% base-pair mismatch, hybridization washes would be carried out at a temperature of about 51 °C. It is thus within the skill of one in the art to calculate additional hybridization temperatures based on the desired percentage base-pair mismatch, formulae and G/C content disclosed herein. For example, it is appreciated by one skilled in the art that as the nucleic acid molecule to be tested for hybridization against nucleic acid molecules of the present invention having sequences specified herein becomes longerthan 150 nucleotides, the T_m for a hybridization reaction allowing up to 30% base-pair mismatch will not vary significantly from 51 °C.

Furthermore, it is known in the art that there are commercially available computer programs for determining the degree of similarity between two nucleic acid sequences. These computer programs include various known methods to determine the percentage identity and the number and length of gaps between hybrid nucleic acid molecules. Preferred methods to determine the percent identity among amino acid sequences and also among nucleic acid sequences include analysis using one or more of the commercially available computer programs designed to compare and analyze nucleic acid or amino acid sequences. These computer programs include, but are not limited to, GCG™ (available from Genetics Computer Group, Madison, WI), DNAsis™ (available from Hitachi Software, San Bruno, CA) and MacVector™ (available from the Eastman Kodak Company, New Haven, CT). A preferred method to determine percent identity among amino acid sequences and also among nucleic acid sequences includes using the Compare function by maximum matching within the program DNAsis Version 2.1 using default parameters.

One embodiment of a Bartonella Bh83 protein of the present invention is a fusion protein that includes a Bartonella Bh83 protein-containing domain attached to one or more fusion segments. Suitable fusion segments for use with the present invention include, but are not limited to, segments that can: enhance a protein's stability; act as an immunopotentiator to enhance an immune response against a Bartonella Bh83 protein; and/or assist in purification of a Bartonella Bh83 protein (e.g., by affinity chromatography). A suitable fusion segment can be a domain of any size that has the desired function (e.g., imparts increased stability, imparts increased immunogenicity to a protein, and/or simplifies purification of a protein). Fusion segments can be joined to amino and/or carboxyl termini of the Bartonella Bh83-containing domain of the protein and can be susceptible to cleavage in order to enable straight-forward recovery of a Bartonella Bh83 protein. Fusion proteins are preferably produced by culturing a recombinant cell transformed with a fusion nucleic acid molecule that encodes a protein including the fusion segment attached to either the carboxyl and/or amino terminal end of a Bartonella Bh83-containing domain. Preferred fusion segments include a metal binding domain (e.g., a poly-histidine segment); an immunoglobulin binding domain (e.g., Protein A; Protein G; T cell; B cell; Fc receptor or complement protein antibody-binding domains); a sugar binding domain (e.g., a maltose binding domain); and/or a "tag" domain (e.g., at least a portion of β-galactosidase, a strep tag peptide, a T7 tag peptide, a Flag™ peptide, or other domains that can be purified using compounds that bind to the domain, such as monoclonal antibodies). More preferred fusion segments include metal binding domains, such as a poly-histidine segment; a maltose binding domain; a strep tag peptide, such as that available from Biometra in Tampa, FL; and an S10 peptide. In another embodiment, a Bartonella Bh83 protein of the present invention also includes at least one additional protein segment that is capable of protecting an animal from one or more diseases. Such a multivalent protective protein can be produced by culturing a cell transformed with a nucleic acid molecule comprising two or more nucleic acid domains joined together in such a manner that the resulting nucleic acid molecule is expressed as a multivalent protective compound containing at least two protective compounds capable of protecting an animal from diseases caused, for example, by at least one infectious agent. Examples of multivalent protective compounds include, but are not limited to, a Bartonella Bh83 protein of the present invention attached to one or more compounds protective against one or more other infectious agents, preferably an agent that infects cats, such as, but not limited to: viruses (e.g., adenoviruses, caliciviruses, coronaviruses, distemper viruses, hepatitis viruses, herpesviruses, immunodeficiency viruses, infectious peritonitis viruses, leukemia viruses, oncogenic viruses, panleukopenia viruses, papilloma viruses, parainfluenza viruses, parvoviruses, rabies viruses, and reoviruses, as well as other cancer- causing or cancer-related viruses); bacteria (e.g., Actinomyces, Bacillus, Bacteroides, Bordetella, Bartonella, Borrelia, Brucella, Campylobacter, Capnocytophaga, Clostridium, Corynebacterium, Coxiella, Dermatophilus, Enterococcus, Ehriichia, Escherichia, Francisella, Fusobacterium, Haemobartonella, Helicobacter, Klebsiella, L-form bacteria, Leptospira, Listeria, Mycobacteria, Mycoplasma, Neorickettsia, Nocardia, Pasteurella, Peptococcus, Peptostreptococcus, Proteus, Pseudomonas, Rickettsia, Rochalimaea, Salmonella, Shigella, Staphylococcus, Streptococcus, and Yersinia; fungi and fungal-related microorganisms (e.g., Absidia, Acremonium, Alternaria, Aspergillus, Basidiobolus, Bipolaris, Blastomyces, Candida, Chlamydia, Coccidioides, Conidiobolus, Cryptococcus, Curvalaria, Epidermophyton, Exophiala, Geotrichum, Histoplasma, Madurella, Malassezia, Microsporum, Moniliella, Mortierella, Mucor, Paecilomyces, Penicillium, Phialemonium, Phialophora, Prototheca, Pseudallescheria, Pseudomicrodochium, Pythium, Rhinosporidium, Rhizopus, Scolecobasidium, Sporothrix, Stemphylium, Trichophyton, Trichosporon, and Xylohypha; and other parasites (e.g., Babesia, Balantidium, Besnoitia, Cryptosporidium, Eimeria, Encephalitozoon, Entamoeba, Giardia, Hammondia, Hepatozoon, Isospora, Leishmania, Microsporidia, Neospora, Nosema, Pentatrichomonas, Plasmodium, Pneumocystis, Sarcocystis, Schistosoma, Theileria, Toxoplasma, and Trypanosoma. The present invention also includes mimetopes of Bartonella Bh83 proteins of the present invention. As used herein, a mimetope of a Bartonella Bh83 protein of the present invention refers to any compound that is able to mimic the activity of such a Bartonella Bh83 protein, often because the mimetope has a structure that mimics the particular Bartonella Bh83 protein. Mimetopes can be, but are not limited to: peptides that have been modified to decrease their susceptibility to degradation such as all-D retro peptides; anti- idiotypic and/or catalytic antibodies, or fragments thereof; non-proteinaceous immunogenic portions of an isolated protein (e.g., carbohydrate structures); and synthetic or natural organic molecules, including nucleic acids. Such mimetopes can be designed using computer-generated structures of proteins of the present invention. Mimetopes can also be obtained by generating random samples of amplification or cloning) or chemical synthesis. Examples of isolated Bartonella Bh83 nucleic acid molecules include a nucleic acid molecule encoding a β. henselae Bh83 protein of 83 kDa as well as homologues thereof, such as natural allelic variants, nucleic acid molecules of other Bartonella species that encode proteins that cross-react with an antibody against a B. henselae Bh83 protein of 83 kDa, and nucleic acid molecules modified by nucleotide insertions, deletions, substitutions, and/or inversions of any of the foregoing nucleic acid molecules in a manner such that the modifications do not substantially interfere with the nucleic acid molecule's ability to encode a Bartonella Bh83 protein of the present invention. Also included are nucleic acid molecules having a sequence complementary to a nucleic acid sequence encoding such Bartonella Bh83 nucleic acid molecules. A nucleic acid sequence complement refers to the nucleic acid sequence of the nucleic acid strand that is complementary to (i.e., can form a double helix with) the cited nucleic acid molecule, which can easily be determined by those skilled in the art.

As used herein, an allelic variant of a Bartonella Bh83 gene is a gene that occurs at essentially the same locus (or loci) in the genome as a Bartonella Bh83 gene, but which, due to natural variations caused by, for example, mutation or recombination, has a similar but not identical sequence. Because natural selection typically selects against alterations that affect function, allelic variants (i.e., alleles corresponding to, or of, cited nucleic acid sequences) usually encode proteins having similar activity to that of the protein encoded by the gene to which they are being compared. Allelic variants of genes or nucleic acid molecules can also comprise alterations in the 5' or 3' untranslated regions of the gene (e.g., in regulatory control regions), or can involve alternative splicing of a nascent transcript, thereby bringing alternative exons into juxtaposition. Allelic variants are well known to those skilled in the art and would be expected to occur naturally within a given Bartonella species such as B. henselae or β. quintana.

A Bartonella Bh83 nucleic acid molecule homologue can be produced using a number of methods known to those skilled in the art, see, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring

- 15 - Harbor Labs Press; and Ausubel et al., "Current Protocols in Molecular Biology," John Wiley and Sons, New York 1987 (updated quarterly). For example, nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis and recombinant DNA techniques such as site- directed mutagenesis, chemical treatment, restriction enzyme cleavage, ligation of nucleic acid fragments, PCR amplification, synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules, and combinations thereof. Nucleic acid molecule homologues can be selected by hybridization with a Bartonella Bh83 nucleic acid molecule or by screening the function of a protein encoded by the nucleic acid molecule (e.g., ability to elicit an immune response against at least one epitope of a Bartonella Bh83 protein).

An isolated nucleic acid molecule of the present invention can include a nucleic acid sequence that encodes at least one Bartonella Bh83 protein of the present invention, examples of such proteins being disclosed herein. Although the phrase "nucleic acid molecule" primarily refers to the physical nucleic acid molecule and the phrase "nucleic acid sequence" primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a Bartonella Bh83 protein.

A preferred nucleic acid molecule of the present invention, when administered to an animal, is capable of protecting that animal from infection with a Bartonella bacterium, and preferably from infection with B. henselae or β. quintana which can lead to cat scratch disease, at least in certain animals known to those skilled in the art. As will be disclosed in more detail below, such a nucleic acid molecule can be, or encode, an antisense RNA, a molecule capable of triple helix formation, a ribozyme, or other nucleic acid-based drug compound. In additional embodiments, a nucleic acid molecule of the present invention can encode a protective protein (e.g., a Bartonella Bh83 protein of the present invention), the nucleic acid molecule being delivered to the animal, for example,

- 16 by direct injection (i.e, as a genetic vaccine) or in a vehicle such as a recombinant virus vaccine or a recombinant cell vaccine.

In another embodiment, a preferred fiat one/Va Bh83 nucleic aid molecule of the present invention comprises an apparently full-length Bartonella Bh83 coding region, i.e., the preferred nucleic acid molecule encodes an apparently full-length Bartonella Bh83 protein.

Knowing the nucleic acid sequences of Bartonella Bh83 nucleic acid molecules of the present invention allows one skilled in the art to, for example, (a) make copies of those nucleic acid molecules, (b) obtain nucleic acid molecules including at least a portion of such nucleic acid molecules (e.g., nucleic acid molecules including full-length genes, full-length coding regions, regulatory control sequences, truncated coding regions), and (c) obtain other Bartonella Bh83 nucleic acid molecules. Such nucleic acid molecules can be obtained in a variety of ways including screening appropriate expression libraries with antibodies of the present invention; traditional cloning techniques using oligonucleotide probes of the present invention to screen appropriate libraries; and PCR amplification of appropriate libraries or DNA using oligonucleotide primers of the present invention. Preferred libraries to screen or from which to amplify nucleic acid molecules include Bartonella, and more preferably β. henselae and/or β. quintana cDNA libraries as well as genomic DNA libraries. Similarly, preferred DNA sources from which to amplify nucleic acid molecules include Bartonella, and more preferably ^β. henselae and/or β. quintana cDNA and genomic DNA. Techniques to clone and amplify genes are disclosed, for example, in Sambrook et al., ibid, and Ausubel et al., ibid. The present invention also includes nucleic acid molecules that are oligonucleotides capable of hybridizing, under stringent hybridization conditions, with complementary regions of other, preferably longer, nucleic acid molecules of the present invention such as those comprising Bartonella Bh83 nucleic acid molecules. Oligonucleotides of the present invention can be RNA, DNA, or derivatives of either. The minimum size of such oligonucleotides is the size required for formation of a stable hybrid between an oligonucleotide and a

- 17 - complementary sequence on a nucleic acid molecule of the present invention. The present invention includes oligonucleotides that can be used as, for example, probes to identify nucleic acid molecules, primers to produce nucleic acid molecules, or therapeutic reagents to inhibit Bartonella Bh83 protein production or activity (e.g., as antisense-, triplex formation-, ribozyme- and/or RNA drug-based reagents). The present invention also includes the use of such oligonucleotides to protect animals from disease using one or more of such technologies. Appropriate oligonucleotide-containing therapeutic compositions can be administered to an animal using techniques known to those skilled in the art.

One embodiment of the present invention includes a recombinant vector, which includes at least one isolated nucleic acid molecule of the present invention, inserted into any vector capable of delivering the nucleic acid molecule into a host cell. Such a vector contains heterologous nucleic acid sequences, that is nucleic acid sequences that are not naturally found adjacent to nucleic acid molecules of the present invention and that preferably are derived from a species other than the species from which the nucleic acid molecule(s) are derived. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid. Recombinant vectors can be used in the cloning, sequencing, and/or otherwise manipulating of Bartonella Bh83 nucleic acid molecules of the present invention.

One type of recombinant vector, referred to herein as a recombinant molecule, comprises a nucleic acid molecule of the present invention operatively linked to an expression vector. The phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell. As used herein, an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified nucleic acid molecule. Preferably, the expression vector is also capable of replicating within the host cell. Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids. Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells of the present invention, including in bacterial, fungal, parasite, insect, other animal, and plant cells. Preferred expression vectors of the present invention can direct gene expression in bacterial, yeast, insect and mammalian cells. In particular, expression vectors of the present invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of nucleic acid molecules of the present invention. In particular, recombinant molecules of the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art. Preferred transcription control sequences include those which function in bacterial, yeast, or insect and mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy- pro, omp/lpp, rrnB, bacteriophage lambda (such as lambda p_L and lambda p_R and fusions that include such promoters), bacteriophage T7, T7/ac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01 , metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoter, antibiotic resistance gene, baculovirus, Heliothiszea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus, adenovirus, cytomegalovirus (such as immediate early promoter), simian virus 40, retrovirus, actin, retroviral long terminal repeat, Rous sarcoma virus, heat shock, phosphate and nitrate transcription control sequences as well as other sequences capable of controlling gene expression in prokaryotic or eukaryotic cells. Additional suitable transcription control sequences include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by

- 19 - molecules, such as oligonucleotides, peptides or other organic molecules, and screening such samples by affinity chromatography techniques using the corresponding binding partner.

The present invention also includes Bartonella Bh83 nucleic acid molecules that encode Bartonella Bh83 proteins. Bartonella Bh83 proteins of the present invention can be used to create primers which can be used to screen Bartonella cDNA expression libraries and/or genomic DNA libraries prepared using procedures well known to those skilled in the art. Screening permits identification of clones encoding Bartonella Bh83 proteins As used herein, a Bartonella Bh83 nucleic acid molecule includes nucleic acid sequences related to a natural Bartonella Bh83 gene, and, preferably, to either a B. henselae Bh83 gene or a B. quintana Bh83 gene. As used herein, a Bartonella Bh83 gene includes all regions such as regulatory regions that control production of the Bartonella Bh83 protein encoded by the gene (such as, but not limited to, transcription, translation or post-translation control regions) as well as the coding region itself, and any introns or non-translated coding regions. As used herein, a gene that "includes" or "comprises" a sequence can include that sequence in one contiguous array, or can include the sequence as fragmented exons. As used herein, the term "coding region" refers to a continuous linear array of nucleotides that translates into a protein. A full-length coding region is that coding region that is translated into a full-length, i.e., a complete protein as would be initially translated in its natural millieu, prior to any post-translational modifications.

In accordance with the present invention, an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subjected to human manipulation) and can include DNA, RNA, or derivatives of either DNA or RNA. As such, "isolated" does not reflect the extent to which the nucleic acid molecule has been purified. An isolated Bartonella Bh83 nucleic acid molecule of the present invention, including a homologue thereof, can be isolated from its natural source or produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR)

- 14 - interferons or interleukins). Transcription control sequences of the present invention can also include naturally occurring transcription control sequences naturally associated with Bartonella, such as β. henselae and/or β. quintana transcription control sequences. Recombinant molecules of the present invention can also (a) contain secretory signals (i.e., signal segment nucleic acid sequences) to enable an expressed Bartonella Bh83 protein of the present invention to be secreted from the cell that produces the protein and/or (b) contain fusion sequences which lead to the expression of nucleic acid molecules of the present invention as fusion proteins. Examples of suitable signal segments include any signal segment capable of directing the secretion of a protein of the present invention. Preferred signal segments include, but are not limited to, tissue plasminogen activator (t- PA), interferon, interleukin, growth hormone, histocompatibility and viral envelope glycoprotein signal segments. Suitable fusion segments encoded by fusion segment nucleic acids are disclosed herein. In addition, a nucleic acid molecule of the present invention can be joined to a fusion segment that directs the encoded protein to the proteosome, such as a ubiquitin fusion segment. Eukaryotic recombinant molecules can also include intervening and/or untranslated sequences surrounding and/or within the nucleic acid sequences of nucleic acid molecules of the present invention.

Another embodiment of the present invention includes a recombinant cell comprising a host cell transformed with one or more recombinant molecules of the present invention. Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell can remain unicellular or can grow into a tissue, organ or a multicellular organism. It is to be noted that a cell line refers to any recombinant cell of the present invention that is not a transgenic animal. Transformed nucleic acid molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i e , recombinant) cell in such a manner that their ability to be expressed is retained Preferred nucleic acid molecules with which to transform a cell include Bartonella Bh83 nucleic acid molecules disclosed herein Suitable host cells to transform include any cell that can be transformed with a nucleic acid molecule of the present invention Host cells can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule (e g , nucleic acid molecules encoding one or more proteins of the present invention and/or other proteins useful in the production of multivalent vaccines) Host cells of the present invention either can be endogenously (i e , naturally) capable of producing Bartonella Bh83 proteins of the present invention or can be capable of producing such proteins after being transformed with at least one nucleic acid molecule of the present invention Host cells of the present invention can be any cell capable of producing at least one protein of the present invention, and include bacterial, fungal (including yeast), parasite (including helminth, protozoa and ectoparasite), other insect, other animal and plant cells Preferred host cells include bacterial, mycobacteπal, yeast, insect and mammalian cells More preferred host cells include Salmonella, Eschenchia, Bacillus, Listena, Saccharomyces Spodoptera, Mycobacteria, Tnchoplusia, BHK (baby hamster kidney) cells, MDCK cells (Madin-Darby canine kidney cell line), CRFK cells (Crandell feline kidney cell line), CV-1 cells (African monkey kidney cell line used, for example, to culture raccoon poxvirus), COS (e g , COS-7) cells, and Vero cells Particularly preferred host cells are Eschenchia coll, including E coli K-12 derivatives, Salmonella typhi, Salmonella typhimunum, including attenuated strains such as UK-1 _χ3987 and SR-11 _χ4072, Spodoptera frugiperda, Tnchoplusia ni, BHK cells, MDCK cells, CRFK cells, CV-1 cells, COS cells, Vero cells, and non-tumoπgenic mouse myoblast G8 cells (e g , ATCC CRL 1246) Additional appropriate mammalian cell hosts include other kidney cell lines, other fibroblast cell lines (e g , human, munne or chicken embryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovary cells, mouse NIH/3T3 cells, LMTK³¹ cells and/or HeLa cells. In one embodiment, the proteins can be expressed as heterologous proteins in myeloma cell lines employing immunoglobulin promoters.

A recombinant cell is preferably produced by transforming a host cell with one or more recombinant molecules, each comprising one or more nucleic acid molecules of the present invention operatively linked to an expression vector containing one or more transcription control sequences, examples of which are disclosed herein.

A recombinant cell of the present invention includes any cell transformed with at least one of any nucleic acid molecule of the present invention. Suitable and preferred nucleic acid molecules as well as suitable and preferred recombinant molecules with which to transfer cells are disclosed herein.

Recombinant cells of the present invention can also be co-transformed with one or more recombinant molecules including Bartonella Bh83 nucleic acid molecules encoding one or more proteins of the present invention and one or more other nucleic acid molecules encoding other protective compounds, as disclosed herein (e.g., to produce multivalent vaccines).

Recombinant DNA technologies can be used to improve expression of transformed nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within a host cell, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications. Recombinant techniques useful for increasing the expression of nucleic acid molecules of the present invention include, but are not limited to, operatively linking nucleic acid molecules to high-copy number plasmids, integration of the nucleic acid molecules into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of nucleicacid molecules of the present invention to correspond to the codon usage of the host cell, deletion of sequences that destabilize transcripts, and use of control signals that temporally separate recombinant cell growth from recombinant enzyme production during fermentation. The activity of an expressed recombinant protein of the present invention can be improved by fragmenting, modifying, or derivatizing nucleic acid molecules encoding such a protein. Isolated Bartonella Bh83 proteins of the present invention can be produced in a variety of ways, including production and recovery of natural proteins, production and recovery of recombinant proteins, and chemical synthesis of the proteins. In one embodiment, an isolated protein of the present invention is produced by culturing a cell capable of expressing the protein under conditions effective to produce the protein, and recovering the protein. A preferred cell to culture is a recombinant cell of the present invention. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. An effective medium refers to any medium in which a cell is cultured to produce a Bartonella Bh83 protein of the present invention. Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art. Examples of suitable conditions are included in the Examples section.

Depending on the vector and host system used for production, resultant proteins of the present invention can either remain within the recombinant cell; be secreted into the fermentation medium; be secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or be retained on the outer surface of a cell or viral membrane.

In one embodiment, Bartonella Bh83 proteins of the present invention can be produced from natural sources. Bartonella can be propagated by bacteriological methods and purified therefrom using methods well-known to those skilled in the art, examples of which are disclosed herein. For example, Bartonella can be grown upon a eukaryotic cell monolayer. Suitable eukaryotic cells upon which to grow Bartonella include monkey cells, human cells, mouse cells, cat cells and insect cells, with monkey cells being preferred. Preferred monkey cells upon which to grow Bartonella include Vero cells. Bartonella can also be grown on solid agar, such as on standard bacteriological agar plates, dishes or trays, for example, heart infusion agar supplemented with rabbit blood (HIA rabbit blood agar). Bartonella can also be growth in bacteriological broth suspension culture, forexample, in supplemented Brucella broth. Bartonella can be propagated at a variety of temperatures. A preferable growth temperature is from about 30°C to about 39°C. More preferable is a growth temperature from about 32°C to about 37°C.

The phrase "recovering the protein", as well as similar phrases, refers to collecting the whole fermentation medium containing the protein and need not imply additional steps of separation or purification. Proteins of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization. Proteins of the present invention are preferably retrieved in "substantially pure" form. As used herein, "substantially pure" refers to a purity that allows for the effective use of the protein as a therapeutic composition or diagnostic. A therapeutic composition for animals, for example, should exhibit no substantial toxicity and preferably should be capable of stimulating the production of antibodies in a treated animal.

The present invention also includes isolated (i.e., removed from their natural milieu) antibodies that selectively bind to a Bartonella Bh83 protein of the present invention or a mimetope thereof (e.g., anW-Bartonella Bh83 antibodies). As used herein, the terms "selectively binds to" or "is immunoreactive with" a Bartonella Bh83 protein refers to the ability of antibodies of the present invention to preferentially bind to specified proteins and mimetopes thereof of the present invention. Binding can be measured using a variety of methods standard in the art including enzyme immunoassays (e.g., ELISA), immunoblot assays, etc.; see, for example, Sambrook et al., ibid., and Harlow, et al., 1988, Antibodies, a Laboratory Manual, Cold Spring Harbor Labs Press; Ausubel et al., "Current Protocols in Molecular Biology," John Wiley and Sons, New York 1987 (updated quarterly); and Harlow et al., ibid. An ant\-Bartonella Bh83 antibody of the present invention preferably selectively binds to a Bartonella Bh83 protein in such a way as to inhibit the function of that protein. Isolated antibodies of the present invention can include antibodies in serum, or antibodies that have been purified to varying degrees. Antibodies of the present invention can be polyclonal or monoclonal, or can be functional equivalents such as antibody fragments and genetically-engineered antibodies including single chain antibodies or chimeric antibodies that can bind to one or more epitopes.

A preferred method to produce antibodies of the present invention includes (a) administering to an animal an effective amount of a protein, peptide or mimetope thereof of the present invention to produce the antibodies and (b) recovering the antibodies. In another method, antibodies of the present invention are produced recombinantly using techniques as heretofore disclosed to produce Bartonella Bh83 proteins of the present invention. Antibodies raised against defined proteins or mimetopes can be advantageous because such antibodies are not substantially contaminated with antibodies against other substances that might otherwise cause interference in a diagnostic assay or side effects if used in a therapeutic composition.

Antibodies of the present invention have a variety of potential uses that are within the scope of the present invention. For example, such antibodies can be used (a) as therapeutic compounds to passively immunize an animal in order to protect the animal from Bartonella infection susceptible to treatment by such antibodies, (b) as reagents in assays to detect Bartonella infection, and preferably β. henselae ot B. quintana infection, and/or (c) as tools to screen expression libraries and/or to recover desired proteins of the present invention from a mixture of proteins and other contaminants. Furthermore, antibodies of the present invention can be used to target cytotoxic agents to Bartonella in orderto directly kill bacteria. Targeting can be accomplished by conjugating (i.e., stably joining) such antibodies to the cytotoxic agents using techniques known to those skilled in the art. Suitable cytotoxic agents are known to those skilled in the art.

One embodiment of the present invention is a therapeutic composition that, when administered to an animal in an effective manner, is capable of protecting that animal from Bartonella infection, and preferably from B. henselae or β. quintana infection. Therapeutic compositions of the present invention include at least one of the following protective compounds: an isolated Bartonella Bh83 protein or a mimetope thereof, an isolated Bartonella Bh83 nucleic acid molecule, an isolated antibody that selectively binds to a Bartonella Bh83 protein, an inhibitor of Bartonella Bh83 function identified by its ability to bind to a Bartonella Bh83, and a mixture thereof (i.e. , combination of at least two of the compounds). As used herein, a protective compound refers to a compound that, when administered to an animal in an effective manner, is able to treat, ameliorate, and/or prevent Bartonella infection and preferably from β. henselae orβ. quintana infection. Examples of proteins, nucleicacid molecules, antibodies and inhibitors of the present invention are disclosed herein.

The present invention also includes a therapeutic composition comprising at least one Bartonella Bh83-based compound of the present invention in combination with at least one additional compound protective against one or more infectious agents. Examples of such compounds and infectious agents are disclosed herein.

A therapeutic composition of the present invention can be used in a method to protect a subject animal from Bartonella infection, and preferably from β. henselae or B. quintana infection, by administering the therapeutic composition to that animal. The therapeutic composition can also be used in a method to protect a subject animal, e.g., a susceptible human, from Bartonella infection and preferably from B. henselae or B. quintana infection by administering the therapeutic composition to a carrier animal, e.g., a domestic cat in proximity with the subject animal, thereby preventing bacteremia in the carrier animal and subsequent transmission to the subject animal. Therapeutic compositions of the present invention can be administered to any animal susceptible to such therapy, preferably to mammals, and more preferably to felids and primates. Even more preferred animals to protect against B. henselae infection include wild cats, domestic cats and humans.

Therapeutic compositions of the present invention can be formulated in an excipient that the animal to be treated can tolerate. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, ortriglycerides can also be used. Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal, o-cresol, formalin and benzyl alcohol. Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient can comprise dextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration. In one embodiment of the present invention, a therapeutic composition can include an adjuvant. Adjuvants are agents that are capable of enhancing the immune response of an animal to a specific antigen. Suitable adjuvants include, but are not limited to, cytokines, chemokines, and compounds that induce the production of cytokines and chemokines (e.g., granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), colony stimulating factor (CSF), erythropoietin (EPO), interleukin 2 (IL-2), ιnterleukιn-3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 10 (IL-10), interleukin 12 (IL-12), interferon gamma, interferon gamma inducing factor I (IGIF), transforming growth factor beta, RANTES (regulated upon activation, normal T-cell expressed and presumably secreted), macrophage inflammatory proteins (e g , MIP-1 alpha and MIP-1 beta), flt-3 ligand, and Leishmania elongation initiating factor (LEIF)), bacterial components (e g , endotoxins, in particular superantigens, exotoxins and cell wall components), aluminum-based salts, calcium-based salts, silica, polynucleotides, toxoids, serum proteins, viral coat proteins, block copolymer adjuvants (e g , Hunter's Titermax™ adjuvant (Vaxcel™, Inc Norcross, GA), Ribi adjuvants (Ribi ImmunoChem Research, Inc , Hamilton, MT), and saponins and their derivatives (e g , Quil A (Superfos Biosector A/S, Denmark) Protein adjuvants of the present invention can be delivered in the form of the protein themselves or of nucleic acid molecules encoding such proteins using the methods described herein

In one embodiment of the present invention, a therapeutic composition can include a carrier Carriers include compounds that increase the half-life of a therapeutic composition in the treated animal Suitable carriers include, but are not limited to, polymeric controlled release vehicles, biodegradable implants, hposomes, bacteria, viruses, other cells, oils, esters, and glycols

One embodiment of the present invention is a controlled release formulation that is capable of slowly releasing a composition of the present invention into an animal As used herein, a controlled release formulation comprises a composition of the present invention in a controlled release vehicle Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, hposomes, lipospheres, and transdermal delivery systems Other controlled release formulations of the present invention include liquids that, upon administration to an animal, form a solid or a gel in situ. Preferred controlled release formulations are biodegradable (i.e., bioerodible).

A preferred controlled release formulation of the present invention is capable of releasing a composition of the present invention into the blood of the treated animal at a constant rate sufficient to attain therapeutic dose levels of the composition to protect an animal from Bartonella infection and preferably from β. henselae or B. quintana infection. The therapeutic composition is preferably released over a period of time ranging from about 1 to about 12 months. A controlled release formulation of the present invention is capable of effecting a treatment preferably for at least about 1 month, more preferably for at least about 3 months, even more preferably for at least about 6 months, even more preferably for at least about 9 months, and even more preferably for at least about 12 months.

Therapeutic compositions of the present invention can be administered to animals prior to infection in order to prevent infection and/or can be administered to animals after infection in order to treat Bartonella infection and preferably B. henselae or B. quintana infection. For example, proteins, mimetopes thereof, and antibodies thereof can be used as immunotherapeutic agents. Acceptable protocols to administer therapeutic compositions in an effective manner include individual dose size, number of doses, frequency of dose administration, and mode of administration. Determination of such protocols can be accomplished by those skilled in the art. A suitable single dose is a dose that is capable of protecting an animal from disease when administered one or more times over a suitable time period. For example, a preferred single dose of a protein, mimetope or antibody therapeutic composition is from about 1 microgram (μg) to about 10 milligrams (mg) ofthe therapeutic composition per kilogram body weight of the animal. Booster vaccinations can be administered from about 2 weeks to several years after the original administration. Booster administrations preferably are administered when the immune response of the animal becomes insufficient to protect the animal from disease. A preferred administration schedule is one in which from about 10 μg to about 1 mg of the therapeutic composition per kg body weight of the animal is administered from about one to about two times over a time period of from about 2 weeks to about 12 months Modes of administration can include, but are not limited to, subcutaneous, intradermal, intravenous, intranasal, oral, transdermal and intramuscular routes

According to one embodiment, a nucleic acid molecule of the present invention can be administered to an animal in a fashion to enable expression of that nucleic acid molecule into a protective protein or protective RNA (e g , antisense RNA, ribozyme, triple helix forms or RNA drug) in the animal Nucleic acid molecules can be delivered to an animal in a variety of methods including, but not limited to, (a) administering a naked (i e , not packaged in a viral coat or cellular membrane) nucleic acid as a genetic vaccine (e g , as naked DNA or RNA molecules, such as is taught, for example in Wolff et al , 1990, Science 247, 1465-1468) or (b) administering a nucleic acid molecule packaged as a recombinant virus vaccine or as a recombinant cell vaccine (i e , the nucleic acid molecule is delivered by a viral or cellular vehicle)

A genetic (i e , naked nucleic acid) vaccine of the present invention includes a nucleic acid molecule of the present invention and preferably includes a recombinant molecule of the present invention that preferably is replication, or otherwise amplification, competent A genetic vaccine of the present invention can comprise one or more nucleic acid molecules of the present invention in the form of, for example, a dicistronic recombinant molecule Preferred genetic vaccines include at least a portion of a viral genome (i e , a viral vector) Preferred viral vectors include those based on alphaviruses, poxviruses, adenoviruses, herpesviruses, picornaviruses, and retroviruses, with those based on alphaviruses (such as sindbis or Semliki forest virus), species-specific herpesviruses and poxviruses being particularly preferred Any suitable transcription control sequence can be used, including those disclosed as suitable for protein production Particularly preferred transcription control sequences include cytomegalovirus immediate early (preferably in conjunction with Intron- A), Rous sarcoma virus long terminal repeat, and tissue-specific transcription control sequences, as well as transcription control sequences endogenous to viral vectors if viral vectors are used. The incorporation of a "strong" polyadenylation signal is also preferred.

Genetic vaccines of the present invention can be administered in a variety of ways, with intramuscular, subcutaneous, intradermal, transdermal, intranasal and oral routes of administration being preferred. A preferred single dose of a genetic vaccine ranges from about 1 nanogram (ng) to about 600 μg, depending on the route of administration and/or method of delivery, as can be determined by those skilled in the art. Suitable delivery methods include, for example, by injection, as drops, aerosolized and/ortopically. Genetic vaccines of the present invention can be contained in an aqueous excipient (e.g., phosphate buffered saline) alone or in a carrier (e.g., lipid-based vehicles).

A recombinant virus vaccine of the present invention includes a recombinant molecule of the present invention that is packaged in a viral coat and that can be expressed in an animal after administration. Preferably, the recombinant molecule is packaging- or replication-deficient and/or encodes an attenuated virus. A number of recombinant viruses can be used, including, but not limited to, those based on alphaviruses, poxviruses, adenoviruses, herpesviruses, picornaviruses, and retroviruses. Preferred recombinant virus vaccines are those based on alphaviruses (such as Sindbis virus), raccoon poxviruses, species-specific herpesviruses and species-specific poxviruses. An example of methods to produce and use alphavirus recombinant virus vaccines are disclosed in PCT Publication No. WO 94/17813, by Xiong et al., published August 18, 1994, which is incorporated by reference herein in its entirety. When administered to an animal, a recombinant virus vaccine of the present invention infects cells within the immunized animal and directs the production of a protective protein or RNA nucleic acid molecule that is capable of protecting the animal from Bartonella infection and preferably from B. henselae or B. quintana infection. For example, a recombinant virus vaccine comprising a Bartonella Bh83 nucleic acid molecule of the present invention is administered according to a protocol that results in the animal producing a sufficient immune response to protect itself from Bartonella infection and preferably from B. henselae ot B. quintana infection. A preferred single dose of a recombinant virus vaccine of the present invention is from about 1 x 10⁴ to about 1 x 10⁸ virus plaque forming units (pfu) per kilogram body weight of the animal. Administration protocols are similar to those described herein for protein-based vaccines, with subcutaneous, intramuscular, intranasal and oral administration routes being preferred.

A recombinant cell vaccine of the present invention includes recombinant cells of the present invention that express at least one protein of the present invention. Preferred recombinant cells for this embodiment include Salmonella, E. coli, Listeria, Mycobacterium, S. frugiperda, yeast (including Saccharomyces cerevisiae and Pichia pastoris), BHK, CV-1 , myoblast G8, COS (e.g., COS-7), Vero, MDCK and CRFK recombinant cells. Recombinant cell vaccines of the present invention can be administered in a variety of ways but have the advantage that they can be administered orally, preferably at doses ranging from about 10⁸ to about 10¹² cells per kilogram body weight. Administration protocols are similar to those described herein for protein-based vaccines. Recombinant cell vaccines can comprise whole cells, cells stripped of cell walls or cell lysates.

The efficacy of a therapeutic composition of the present invention to protect an animal from Bartonella infection and preferably from B. henselae or β. quintana infection can be tested in a variety of ways including, but not limited to, detection of protective antibodies (using, for example, proteins or mimetopes of the present invention), detection of cellular immunity within the treated animal, or challenge of the treated animal with a Bartonella microorganism to determine whether the treated animal is resistant to infection or disease from that microorganism. In one embodiment, therapeutic compositions can be tested in animal models such as mice. Such techniques are known to those skilled in the art.

One preferred embodiment of the present invention is the use of Bartonella Bh83 proteins, nucleic acid molecules, antibodies and inhibitors of the present invention, to protect an animal from Bartonella infection and preferably from β henselae or B quintana infection Particularly preferred therapeutic compositions include Bartonella Bh83-based therapeutic compositions of the present invention Such compositions include Bartonella Bh83 nucleic acid molecules, Bartonella Bh83 proteins and mimetopes thereof, a nti-βatf onella Bh83 antibodies, and inhibitors of Bartonella Bh83 function Therapeutic compositions are administered to animals in a manner effective to protect the animals from Bartonella infection and preferably from B henselae or B quintana infection Additional protection can be obtained by administering additional protective compounds One therapeutic composition of the present invention includes an inhibitor of βat oπe//a Bh83 function, i e , a compound capable of substantially interfering with the function of a Bartonella Bh83 protein susceptible to inhibition For example, an isolated protein or mimetope thereof is administered in an amount and manner that elicits (i e , stimulates) an immune response that is sufficient, upon interaction with a native Bartonella Bh83, to protect the animal from the disease Similarly, an antibody of the present invention, when administered to an animal in an effective manner, is administered in an amount so as to be present in the animal at a titer that is sufficient, upon interaction of that antibody with a native Bartonella Bh83, to protect the animal from the disease, at least temporarily Oligonucleotide nucleic acid molecules of the present invention can also be administered in an effective manner, thereby reducing expression of Bartonella Bh83 proteins

An inhibitor of Bartonella Bh83 function can be identified using Bartonella Bh83 proteins of the present invention A preferred method to identify a compound capable of inhibiting Bartonella Bh83 activity includes contacting an isolated Bartonella Bh83 with a putative inhibitory compound under conditions in which, in the absence of said compound, said protein has Bartonella Bh83 activity, and determining if said putative inhibitory compound inhibits said activity

It is also within the scope of the present invention to use isolated proteins, mimetopes, nucleic acid molecules and antibodies of the present invention as diagnostic reagents to detect Bartonella infection and preferably B. henselae or β. quintana infection. Methods to use such diagnostic reagents to diagnose Bartonella infection and preferably B. henselae or β. quintana infection are well known to those skilled in the art. Suitable and preferred Bartonella species to detect are those to which therapeutic compositions of the present invention are targeted. Preferred Bartonella species to detect using diagnostic reagents of the present invention are B. henselae and B. quintana.

The following examples are provided for the purposes of illustration and are not intended to limit the scope of the present invention. All references cited in the present specification are hereby incorporated by reference.

Example 1 This example describes the production of whole-cell β henselae and β. quintana antigen preparations for Western blot analysis.

A. Whole-cell agar-grown B. henselae and B. quintana antigen preparations. Agar-grown B. henselae and B. quintana antigen preparations were produced as follows, β. henselae strain Houston- 1 , ATCC #49882, and β. quintana strain OK-90-268, described by Welch et al., 1992, J. Clinical Microbiology, 30:275-280, were independently grown on heart infusion agar supplemented with 5% defibrinated rabbit blood, available from BBL, Cockeysville, MD. Inoculated plates were incubated for 3 to 5 days at 32°C in the presence of 5% CO₂ followed by harvesting by scraping plates with a sterile loop and suspending released cells in brain heart infusion media (BHI), available from BBL. Suspended cells were collected via centrifugation and suspended in phosphate-buffered saline solution (PBS) to obtain a preparation of agar-grown B. henselae at approximately 1 x 10⁸ CFU/ml or a preparation of β. quintana at approximately 1 x 10⁸ CFU/ml, respectively. Colony forming units (CFU) of harvested B. henselae and B. quintana cultures were titrated on blood agar plates before being inactivated by 5 x 10⁵ rads gamma irradiation and stored at -70°C until used.

B. Whole-cell Vero cell-grown B. henselae and B. quintana antigen preparations. Cell culture-derived B. henselae and β. quintana antigen preparations were produced as follows. Vero cells (available as Catalog No. ATCC CRL-1586 from the American Type Culture Collection (ATCC), Rockville, MD) were cultured to near-confluency in 150-cm² tissue culture flasks in medium comprising MEM supplemented with fetal bovine serum (10%), L-glutamine (2 mM), non-essential amino acids (0.1 mM), and HEPES buffer (10 mM) (all components available from Life Technologies, Inc., Gaithersburg, MD). This medium formulation is referred to herein as MEMsuppl. The adherent cultured Vero cells were washed twice with sterile phosphate buffered saline (PBS), and then 10 ml of MEMsuppl was added to each flask. B. henselae strain Houston-1 and β. quintana strain OK-90-268, prepared as described herein, were independently co-cultivated with antibiotic-free Vero cell monolayers by adding about 1 x 10⁶ CFU of Bartonella strains to shake flasks containing Vero cell monolayers. Cell cultures were incubated for 2 to 4 days post inoculation at 32°C in the presence of 5% CO₂ followed by harvesting by gently rocking culture plates with glass beads to detach Vero cells. Detached cultures were inactivated by 5 x 10⁵ rads gamma irradiation and stored at -70°C until used.

Example 2. This example describes the identification of a Bartonella Bh83 protein of the present invention. Whole-cell antigen preparations of Example 1 were tested for protein concentration by a bicicurinic acid (BCA) Protein Reagent Assay, available from Bio-Rad, Hercules, CA and determined to be about 7.5 milligrams of protein per milliliter. Antibody analysis of each antigen preparation was conducted as follows: 125 microliter (μL) aliquots of each whole-cell antigen preparation was centrifuged for 10 minutes at 13,000 rpm. The cell pellet was solubilized and lysed in 1X Tris-Glycine sodium dodecyl sulfate (SDS) sample buffer, available from Novex, San Diego, CA, containing 10% beta-mercaptoethanol for 10 minutes at 100°C. The resulting suspension was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in a 4 to 20% gradient polyacrylamide Tris-Glycine single-well minigel, available from Novex, for 2 hours at 120 volts. A pre-stained broad-range molecular weight protein marker, available from Bio-Rad, was used as a standard. Following SDS-PAGE, proteins were electrophoretically transferred to a 0.45 micrometer (μm) nitrocellulose membrane, available from Bio-Rad, for 2 h hours at 90 volts in transfer buffer, available from Novex. Membranes were blocked overnight at 4°C in PBS containing 0.1 % Tween 20 (PBST) and 5% skim milk. Membranes were subsequently washed four times, 10 minutes per wash, in PBST and then incubated for 1 hour in a 1 :100 dilution of test serum in PBST and 5% milk using a Mini Protean II Multiscreen System, available from Bio-Rad. Membranes were again washed four times, 10 minutes per wash, and then reacted for 1 hour at room temperature with a 1 :5000 dilution of horseradish peroxidase-labeled anti- human immunoglobulin, available from Kirkegaard & Perry, Gaithersburg, MD, diluted in PBST and 10% skim milk. Membranes were again washed four times, 10 minutes per wash, and antigens were detected using a 3,3', 5,5' tetramethyl benzidine (TMB) membrane substrate developer, available from Kirkegaard & Perry. The following horseradish peroxidase-labeled anti-human immunoglobulins were separately tested' anti-lgG (heavy and light chains), anti- IgG.,, anti-lgG₂, anti-lgG₃, anti-lgG₄, anti-lgM, anti-lgE, anti-lgA secretory, and anti-lgA alpha.

A total of sixty-nine human serum samples were used: 54 samples were derived from patients with laboratory diagnosed B. henselae or B. quintana infection, as indicated by indirect fluorescent-antibody assay (IFA) seropositivity to β. henselae or B. quintana antigens; and 15 samples were obtained from patients for whom negative IFA results were obtained.

Western blotting of whole-cell Vero cell-grown and agar-grown B henselae antigen preparations yielded multiple bands ranging in size from about 6 kDa to greater than about 150 kDa. Banding patterns exhibited variability depending upon individual serum reactivity Analysis of differences between sera reactivity to agar-grown and Vero cell-grown B. henselae antigen preparations revealed that Vero cell-grown antigen preparations yielded more numerous bands than agar-grown antigen preparations. However, with regard to β. bense/ae-specific reactivity, differences between antigen preparations were unremarkable, indicating that additional bands observed in Vero cell-grown antigen preparations may have been caused by reactivity against Vero proteins. Despite the variability among reactions with whole-cell B. henselae antigen preparations, an approximately 83-kDa protein, i.e., a B. henselae Bh83 protein of 83 kDa, was immunoreactive with all human sera that were also positive for antigen by IFA analysis. This 83-kDa protein was not immunoreactive with any of the IFA seronegative sera tested by Western blot. Figure 1 shows gels from Western blots containing a subset of tested sera and Table 1 shows IFA results corresponding to sera shown in Figure 1.

Table 1. Human serum specimens received at the Centers for Disease Control and Prevention for diagnosis of CSD by IFA.

Titers are reported as the reciprocal of serum dilution's; 31 is convention for <32. An intense signal at 8192 (the highest dilution tested) is considered >8192. Thus, 8193 is convention for >8192.

Whole-cell β. henselae antigen preparations were also tested for cross- reactivity with human antisera to other bacteria by Western blot. The following human antisera, obtained from the reference serum bank of the U.S. Centers for Disease Control and Prevention (CDC) or the CDC rickettsial zoonoses laboratory stocks were tested for cross-reactivity: Rickettsia rickettsii, Chlamydia group positive sera (CDC#CD0022), Treponeum pallidum (CDC# BS1505 and BS30612), Orientia tsutsugamushi (scrub typhus agent), Fransciscella tularensis (SCD # BS0864), Ehriichia chaffeensis, Mycoplasma pneumoniae (CDC# MS2204), Eschenchia coli and PCR-confirmed β. G/u/n ana-infected human sera. Each serum tested showed binding to 10 to 15 β. henselae bands. Two Rickettsia group antisera demonstrated the least amount of cross- reactivity with β. henselae antigen preparations; in particular, the spotted fever group Rickettsia (Rickettsia rickettsii) yielded only weak activity, with two bands in the 200 kDa region. Treponema pallidum and Chlamydia each reacted strongly to B. henselae antigen preparations in the range of approximately 45 kDa and 75 kDa, respectively. Despite the extensive binding of numerous antigenic proteins, however, none of the cross-reactive bacterial antisera was immunoreactive with the 83-kDa band of B. henselae.

Western blot analysis of human sera reactivity against whole-cell B. henselae antigen preparations indicates a strong IgG reaction of a heterogeneic nature against total antigen, with a specific reaction to the 83-kDa band of B. henselae. To determine which human subclasses of IgG were responsible for these interactions, Western blot assays specific for IgG.,, lgG₂, lgG₃, and lgG₄ were performed. Despite multiple banding patterns in assays detecting total IgG against B. henselae antigen preparations, little B. henselae reactivity was evidenced by lgG₂, lgG₃ or lgG₄. A prominent reaction against B. henselae antigen preparations among the IgG subclasses tested was limited to IgG.,. Immunoblot banding patterns using anti-lgG₁ conjugate were extremely similar to those produced when using anti-total IgG (H & L), suggesting that lgG₁ is the primary IgG subclass induced during B. henselae infection. In addition to analysis of the antibody response at the IgG subclass level,

Western blot analysis was employed to detect differences among the immunoglobuiin isotypes represented in β. henselae antigen positive sera as determined by IFA. B. henselae specific IgA (secretory chain), IgA (alpha chain), IgE, and IgM antibodies were all identified in B. A/ense/ae-positive sera. Both the secretory and alpha chains of IgA demonstrated multi-band binding of whole-cell ^β. henselae antigen in 15 IFA seropositive samples tested. IgA reactivity varied with sera tested, being heterogeneous with similar protein band reactivity as compared with total IgG. Despite the lesser degree of antigen binding, however, the 83-kDa B. henselae protein was immunoreactive with IgA antibodies in all B. frense/ae-positive sera tested. IgM binding in B. henselae antigen preparations was essentially absent, with only two very faint bands in only two of the 25 B. henselae IFA positive sera tested. Additionally, binding of IgE antibodies against β. henselae antigen preparations was absent in 25 B. henselae IFA positive sera tested.

Whole-cell B. henselae and β. quintana antigen preparations have been repeatedly shown to cross-react by IFA, presumably due to close phylogenetic relatedness. The level of cross-reactivity between these two Bartonella species was assessed by Western blot analysis. Separate Western blots were conducted with whole-cell B. henselae and B. quintana antigen preparations using each of β. frense/ae-positive sera, and PCR confirmed B.

sera. Each reaction yielded some degree of cross-reactivity. Sera from PCR- confirmed B. quintana infections reacted with B. henselae and to a lesser extent with β. quintana antigen preparations in Western blots. Included among the B. henselae immunoreactive antigens bound by B. quintana antisera was the 83- kDa β. henselae antigen. However, when B. quintana antigen preparations were screened with either B. henselae- or B. quintana -reactive human sera, reaction to the 83-kDa protein was absent. B. quintana antigen, therefore, appears to lack expression of an 83-kDa antigen per se. Since B. quintana cross-reacts with β. henselae Bh83, without being bound by theory, one might predict that the native β. quintana homologue of β. henselae Bh83 is of a different size. While various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. It is to be expressly understood, however, that such modifications and adaptations are within the scope of the present invention, as set forth in the following claims.

Claims

CLAIMSWhat is claimed is:

1. An isolated Bartonella Bh83 protein comprising at least one epitope that elicits an immune response against a B. henselae Bh83 protein of 83 kDa.

2. The protein of Claim 1 , wherein said protein comprises a B. henselae protein having a molecular weight of about 83 kDa and wherein said protein is immunoreactive with serum from a patient having antibodies against B. henselae.

3. The protein of Claim 1 , wherein said protein is not immunoreactive with serum against an organism selected from the group consisting of: Rickettsia rickettsii, Chlamydia, Treponema pallidum, Orientia tsutsugamushi, Fransciscella tularensis, Ehriichia chaffeensis, Mycoplasma pneumoniae and Escherichia coli.

4. The protein of Claim 2, wherein said protein is not immunoreactive with serum against an organism selected from the group consisting of: Rickettsia rickettsii, Chlamydia, Treponema pallidum, Orientia tsutsugamushi, Fransciscella tularensis, Ehriichia chaffeensis, Mycoplasma pneumoniae and Escherichia coli.

5. The protein of Claim 1 , wherein said protein is immunoreactive with serum from patients having antibodies against B. henselae.

6. The protein of Claim 1 , wherein said protein is immunoreactive with serum from patients having antibodies against β. quintana.

7. The protein of Claim 1 , wherein said Bh83 protein selectively binds to IgG antibodies.

8. The protein of Claim 1 , wherein said Bh83 protein selectively binds to IgA antibodies.

9. The protein of Claim 1 , wherein said protein, when administered to an animal elicits an immune response against a B. henselae Bh83 protein of 83 kDa.

10. The protein of Claim 1 , wherein said protein, when administered to an animal elicits an immune response against a B. henselae Bh83 protein of 83 kDa and protects the animal from Bartonella infection.

11. The protein of Claim 1 , wherein said protein is produced by a process comprising culturing a recombinant cell transformed with a nucleic acid molecule encoding said protein to produce said protein.

12. An isolated nucleic acid molecule that encodes a Bartonella Bh83 protein comprising at least one epitope that elicits an immune response against a β. henselae Bh83 protein of 83 kDa.

13. An isolated antibody that selectively binds to a Bartonella Bh83 protein comprising at least one epitope that elicits an immune response against a β. henselae Bh83 protein of 83 kDa.

14. A recombinant molecule comprising a nucleic acid molecule that encodes a Bartonella Bh83 protein comprising at least one epitope that elicits an immune response against a β. henselae Bh83 protein of 83 kDa and which is operatively linked to a transcription control sequence.

15. A recombinant virus comprising a nucleic acid molecule that encodes a Bartonella Bh83 protein comprising at least one epitope that elicits an immune response against a B. henselae Bh83 protein of 83 kDa.

16. A recombinant cell comprising a nucleic acid molecule that encodes a Bartonella Bh83 protein comprising at least one epitope that elicits an immune response against a β. henselae Bh83 protein of 83 kDa.

17. A method for determining Bartonella infection in an animal suspected of Bartonella infection, said method comprising (1) obtaining serum from the animal, (2) exposing the serum to a Bartonella protein which is immunoreactive with antibodies against β. henselae or B. quintana., and (3) determining whether the Bartonella protein is immunoreactive with the serum.

18. The method of Claim 17, wherein the animal is a domestic cat or a human.

19. A method for preventing Bartonella infection in an animal, said method comprising administering to the animal an effective amount of a Bartonella protein which elicits an immune response against a B. henselae Bh83 protein of 83 kDa and protects the animal from Bartonella infection.

20. The method as defined in Claim 18, wherein the animal is a domestic cat and wherein the animal cannot act as a carrier to transmit Bartonella infection to a human.