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WO2001092535A1 - Operon virb de bartonella henselae et proteines codees par cet operon - Google Patents

Operon virb de bartonella henselae et proteines codees par cet operon Download PDF

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Publication number
WO2001092535A1
WO2001092535A1 PCT/US2000/015465 US0015465W WO0192535A1 WO 2001092535 A1 WO2001092535 A1 WO 2001092535A1 US 0015465 W US0015465 W US 0015465W WO 0192535 A1 WO0192535 A1 WO 0192535A1
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Prior art keywords
seq
nucleotide sequence
nucleic acid
polypeptide
henselae
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Indira Padmalayam
Robert Massung
Kevin Karem
Barbara Baumstark
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Centers of Disease Control and Prevention CDC
US Department of Health and Human Services
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Centers of Disease Control and Prevention CDC
US Department of Health and Human Services
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Priority to AU2000253239A priority Critical patent/AU2000253239A1/en
Priority to EP00938158A priority patent/EP1290177A1/fr
Priority to CA002414125A priority patent/CA2414125A1/fr
Publication of WO2001092535A1 publication Critical patent/WO2001092535A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to Bartonella henselae nucleic acid molecules and polypeptides encoded by those nucleic acid molecules.
  • the present invention also includes therapeutic and diagnostic compositions comprising such nucleic acid molecules and proteins.
  • Bartonella henselae is a Gram negative bacterium within the alpha-2 subgroup of the class Proteobacteria.
  • the genus Bartonella includes eleven species, four of which are recognized as human pathogens (Bass, J. W., et al., "The expanding spectrum of Bartonella infections: I. Bartonellosis and trench fever," The Pediatr. Infect. Dis. J., 16:2 (1997)).
  • B. henselae is the causative agent of cat-scratch disease (CSD), as well as bacillary angiomatosis (BA) and a number of other disease syndromes.
  • CSD cat-scratch disease
  • BA bacillary angiomatosis
  • CSD which occurs primarily in immunocompetent individuals, is typically a benign and self-limiting disease characterized by regional granulomatous lymphadenitis following the scratch or bite of an infected cat. In about 10% of reported CSD cases, the initial symptoms were followed by serious complications involving the central nervous system and other organs (O'Reilly et al., "Acute clinical disease in cats following infection with a pathogenic strain of Bartonella henselae (LSU16)," Infect. Immun. 67: 3066 (1999)).
  • Bacillary angiomatosis (BA) is a vascular proliferative disease that is characterized by cutaneous and subcutaneous vascular lesions.
  • BA was first described in 1983, and was observed to occur predominantly among patients infected with the human immunodeficiency virus (HIV) (Stoler, M. H., et al. "An atypical subcutaneous infection associated with acquired immunodeficiency syndrome.” Am. I. Clin. Pathol. 80: 714 (1983)).
  • HV human immunodeficiency virus
  • B. henselae has been shown to invade human epithelial cells (Batterman et al., "Bartonella henselae and Bartonella quintana adherence to and entry into cultured human epithelial cells.” Infect. Immun. 63:4553 (1995)) and stimulate human endothelial cell proliferation (Conley, T., et al.
  • the virB operon plays a role in the export of factors involved with the virulence of the bacteria. Furthermore, in at least some members of the alpha-2 subgroup, the proteins of the virB operon play a role in interactions with eukaryotic cells and virulence.
  • the proteins encoded by the virB operon form the structural components of the Type IV secretion system that is associated with the export of proteins or nucleic acids (Zupan, J. R., et al., "Assembly of the VirB transport complex for DNA transfer from Agrobacterium tumefaciens to plant cells," Curr. Opin. Microbiol. 1 : 649 (1998); Christie, P.
  • T-DNA (transforming) DNA, referred to as T-DNA, from the bacteria to plant cells
  • pertussis the causative agent of pertussis (whooping cough)
  • the virB operon is implicated in the secretion of the pertussis toxin from the bacterial cell (Weiss, A. A., et al., "Molecular characterization of an operon required for pertussis toxin secretion," Proc. Natl. Acad. Sci. USA, 90: 2970 (1993)).
  • Helicobacter pylori the etiologic agent of chronic, active gastritis and peptic ulcer disease in humans, proteins homologous to several of the VirB proteins from the cag pathogenicity island are involved in the induction of the pro-inflammatory cytokine, IL-8, that is believed to be responsible for the pathogenesis of the ulceration (Censini, S., et al., "cag, a pathogenicity island of Helicobacter pylori, encodes type l-specific and disease-associated virulence factors," Proc. Natl. Acad. Sci. USA, 93: 14648 (1996); Finlay, B. B., et al., 1997).
  • the present invention includes nucleic acids and their encoded polypeptides related to the virB operon of ⁇ . henselae.
  • the invention includes expression vectors containing the nucleic acids and transformed cell lines containing the expression vectors.
  • the invention includes detection methods for ⁇ . henselae infection, and kits for performing these methods.
  • the methods comprise detecting the nucleic acids or polypeptides of the invention, or antibodies against the polypeptides, in patient fluids.
  • the invention includes therapeutic compositions, such as vaccines, for treating and/or preventing Bartonella henselae infection.
  • the invention includes antibodies against the polypeptides encoded by the B. henselae virB operon.
  • the invention includes methods of producing polypeptides encoded by the ⁇ . henselae virB operon.
  • FIG. 1 is the complete nucleic acid sequence of the Bartonella henselae virB operon. Also listed are the amino acid sequences of the open reading frames in the B. henselae virB operon.
  • FIG. 2 is a Western blot of the 17 kDa antigen reacted with CSD- positive human sera, sera from patients with clinical bartonellosis, and controls.
  • Lanes 1-4 serum samples from patients with CSD as confirmed by a positive IFA titer to B. henselae; lanes 5 and 6, serum samples from patients with clinical bartonellosis as confirmed by a positive IFA titer to B. bacilliformis; lanes 7-10: serum samples from controls with negative IFA titers to Bartonella. Approximate molecular weights are shown to the right. The arrow indicates the position of the 17 kDa antigen.
  • FIG. 3 is a schematic representation (not to scale) of the virB region of ⁇ . henselae.
  • the open reading frames are represented as bars.
  • the strategy that was used to isolate the entire virB region is also shown.
  • the current invention consists of isolated nucleic acids related to the virB operon of B. henselae.
  • One embodiment is a an isolated nucleic acid related to the Bartonella henselae virB operon, wherein the isolated nucleic acid comprises a nucleotide sequence of at least 20 nucleotides, preferably at least 23 nucleotides, in length selected from the group consisting of: a) a first nucleotide sequence comprising at least 20, and preferably at least 23, continuous nucleotides of SEQ ID NO: 1, wherein the continuous nucleotide sequence is not from SEQ ID NO: 22; and b) a second nucleotide sequence that is complimentary to the first nucleotide sequence.
  • nucleic acid comprising a nucleotide sequence of at least 50 nucleotides in length selected from the group consisting of: a) a first nucleotide sequence of at least 50 continuous nucleotides of SEQ ID NO: 1 , wherein the nucleotide sequence is other than SEQ ID NO: 5; b) a second nucleotide sequence that is complimentary to the first nucleotide sequence; c) a third nucleotide sequence of a nucleic acid that selectively hybridizes to a nucleic acid with the second nucleotide sequence; and d) a fourth nucleotide sequence that is at least 70% identical to the first nucleotide sequence.
  • the isolated nucleic acid of this embodiment comprises a nucleotide sequence of at least 20, 23, 50, 75, 100, 250, 500, and 1000 nucleotides in length.
  • nucleic acid and nucleic acid molecule primarily refer to the physical nucleic acid molecule and the phrase “nucleic acid sequence” primarily refers to the sequence of nucleotides of the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence of the Bartonella henselae virB operon.
  • isolated nucleic acid molecule(s) is intended a nucleic acid molecule, DNA, or RNA, which has been removed from its native environment.
  • recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention.
  • Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
  • An isolated nucleic acid related to the Bartonella virB operon of the present invention can be isolated from its natural source or produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification or cloning) or chemical synthesis.
  • isolated Bartonella virB operon nucleic acid molecules include a nucleic acid molecule comprising the nucleotide sequence of all or part of SEQ ID NO: 1 , as well as homologues thereof, and natural allelic variants.
  • the present invention includes nucleic acid molecules of other Bartonella species and nucleic acid molecules modified by nucleotide insertions, deletions, substitutions, and/or inversions of the Bartonella virB operon nucleic acid molecules.
  • the complete nucleotide sequence of the virB operon of B. henselae is shown in Figure 1 and listed as SEQ ID NO: 1.
  • a "complementary" nucleic acid sequence 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.
  • the complimentary sequence can easily be determined by those skilled in the art.
  • “Selectively hybridize” in this specification means that two nucleic acids hybridize under stringent conditions.
  • Stringent hybridization conditions are 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, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press (1989), and Meinkoth, et al., Anal. Biochem. 138, 267 (1984).
  • nucleic acid is comprised of one or more modified nucleotides, such as inosine.
  • Stringent hybridization conditions can be defined mathematically. As explained in detail in the cited references, 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).
  • hybrid stability is defined by the dissociation temperature (T d ), which is defined as the temperature at which 50% of the duplexes dissociate.
  • T d dissociation temperature
  • the stability at a standard ionic strength is defined by the following equation:
  • T d 4(G + C) + 2(A + T).
  • T d A temperature of 5°C below T d is used to detect hybridization between perfectly matched molecules.
  • 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. (For example, see Sambrook et al., "Molecular Cloning: A Laboratory Manual," eds. N. Ford and C. Nolan, 2d ed., 11.55 1989). In fact, these procedures can be used to determine the T m of any nucleic acid empirically.
  • T m T m
  • these simple procedures for calculating T m allow one skilled in the art to set the hybridization conditions (e.g., altering the salt concentration, the formamide concentration, or the temperature) so that only nucleic acid hybrids with greater than a specified percent base-pair mismatch will hybridize, (id, at 11.47 and 11.55).
  • T m decreases about 1 °C for each 1% of mismatched base-pairs for hybrids greater than about 150 bp
  • T d decreases about 5°C for each mismatched base-pair for hybrids below about 50 bp.
  • Stringent hybridization conditions are those experimental conditions that will allow about 30% base-pair mismatch (i.e., about 70% identity).
  • 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 given nucleic acid molecule will hybridize with another nucleic acid molecule 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.
  • a labeled nucleic acid molecule typically referred to as a probe
  • Examples of common hybridization reaction techniques include, but are not limited to, the Southern and northern blotting procedures.
  • 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.
  • nucleic acid molecule that hybridizes under stringent hybridization conditions with a natural ⁇ . henselae virB operon nucleic acid molecule of about 150 bp in length
  • the following conditions could preferably be used taking into account that 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 with, for example, a radioactive tag.
  • the initial 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.
  • 20X SSC 17.5.3 gram NaCl and about 88.2 gram sodium citrate in 1 liter of water, pH 7.
  • the skilled artisan would calculate the washing conditions required to allow up to 30% base-pair mismatch.
  • the T m of perfect hybrids would be about 81 °C:
  • hybridization washes would be carried out at a temperature of about 51 °C (i.e., 81 °C - 30°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 longer than 150 nucleotides, the T m for a hybridization reaction allowing up to 30% base-pair mismatch will not vary significantly from 51 °C.
  • the minimum size of an isolated nucleic acid of embodiments defined by hybridization is a size sufficient to form a stable hybrid (i.e., hybridizing under stringent hybridization conditions) with the complementary sequence of a natural ⁇ . henselae virB operon polypeptide-coding nucleic acid.
  • the size of the isolated nucleic acid molecule is dependent on the nucleic acid composition and the percent homology between the natural ⁇ . henselae virB operon nucleic acid and the complementary nucleic acid sequence.
  • henselae virB operon polypeptide-coding nucleic acid 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. Therefore, the minimum size of a nucleic acid molecule defined by hybridization is from about 12 to about 18 nucleotides in length.
  • nucleic acid molecule of the present invention can include a portion of an operon, a portion of a polypeptide-coding sequence, a portion of a gene, an entire polypeptide coding sequence, an entire gene, multiple polypeptide-coding sequences, multiple genes, or an entire operon.
  • the nucleic acids falling within the invention are defined solely in terms of their sequence relatedness to a natural B. henselae virB operon polypeptide- coding nucleic acid molecule, independent of hybridization stringencies.
  • the nucleic acids of the present invention are greater than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical with a nucleic acid related to the ⁇ . henselae virB operon.
  • 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 MacDNAsis Version 1997 using default parameters.
  • nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (e.g., Model 377 from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.99% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.
  • a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the expected amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
  • the present invention is further directed to fragments of the isolated nucleic acid molecules described herein.
  • a fragment of an isolated nucleic acid molecule is intended fragments at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 50 nt in length which are useful as diagnostic probes for B. henselae infection as discussed herein.
  • larger fragments 100, 150, 200, 250, 300, 350, 400, 450, and 500 nt in length are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence of SEQ ID NO: 1.
  • fragments which include 20 or more contiguous bases from the nucleotide sequence of SEQ ID NO: 1.
  • the nucleotide sequence is disclosed in SEQ ID NO: 1.
  • restriction endonuclease cleavage or shearing by sonication could easily be used to generate fragments of various sizes.
  • fragments could be generated synthetically.
  • the nucleic acids of the current invention related to SEQ ID NO: 1 can be used to diagnose B. henselae infection.
  • the nucleic acids can be used as probes to detect related nucleic acids from B.
  • 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. 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 complementary sequence on a nucleic acid molecule of the present invention.
  • the present invention includes oligonucleotides that can be used, for example, as probes to identify nucleic acid molecules, primers to produce nucleic acid molecules, or therapeutic reagents to inhibit Bartonella virB polypeptide 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.
  • an allelic variant of the Bartonella virB operon or polypeptide-coding region therein is an operon or coding region that occurs at essentially the same locus (or loci) in the genome as the Bartonella virB operon or polypeptide-coding region described herein, 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 usually encode proteins having similar activity to that of the protein encoded by the gene to which they are being compared.
  • Allelic variants of operons or nucleic acid molecules can also comprise alterations in the 5' or 3' untranslated regions of a gene located in the operon (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 ⁇ . henselae or B. quintana.
  • Bartonella henselae virB operon nucleic acid molecule can be produced using a number of methods known to those skilled in the art. (See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press).
  • 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 oligonucleotides with modified nucleotide sequences, ligation of oligonucleotides with modified nucleotide sequences, and combinations thereof.
  • 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 oligonucleotides with modified nucleotide sequences, ligation of oligonucleotides with modified nucleotide sequences, and combinations thereof.
  • Nucleic acid molecule homologues can be selected by selective hybridization with all or a portion of a Bartonella henselae virB operon 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 polypeptide encoded by a virB operon protein-coding region).
  • the invention is an isolated nucleic acid that comprises a nucleotide sequence selected from the group consisting of: a) a first nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11 ; b) a second nucleotide sequence of a nucleic acid that selectively hybridizes to a nucleic acid that is fully complementary to the first nucleotide sequence; c) a third nucleotide sequence that is at least 70% identical to the first nucleotide sequence; and d) a fourth nucleotide sequence of a nucleic acid that encodes the polypeptide encoded by any of the first, second, or third nucleotide
  • the isolated nucleic acid comprises the nucleotide sequence of SEQ ID NO: 2. In another preferred embodiment, the isolated nucleic acid comprises the nucleotide sequence of SEQ ID NO: 3. In another preferred embodiment, the isolated nucleic acid comprises the nucleotide sequence of SEQ ID NO: 4. In another preferred embodiment, the isolated nucleic acid comprises the nucleotide sequence of SEQ ID NO: 6. In another preferred embodiment, the isolated nucleic acid comprises the nucleotide sequence of SEQ ID NO: 7. In another preferred embodiment, the isolated nucleic acid comprises the nucleotide sequence of SEQ ID NO: 8. In another preferred embodiment, the isolated nucleic acid comprises the nucleotide sequence of SEQ ID NO: 9. In another preferred embodiment, the isolated nucleic acid comprises the nucleotide sequence of SEQ ID NO: 10. In another preferred embodiment, the isolated nucleic acid comprises the nucleotide sequence of SEQ ID NO: 11.
  • nucleic acids of the present invention with nucleotide sequences related to SEQ ID NOS:2-4 and 6-11 are useful in diagnosing B. henselae infection, as described above for nucleic acids related to SEQ ID NO: 1. Additionally, nucleic acids related to SEQ ID NOS: 2-4 and 6-11 are useful in producing polypeptides of ⁇ . henselae. Such polypeptides can be used, inter alia, in diagnostic assays or therapeutic compositions.
  • the current invention is a recombinant expression vector comprising a nucleic acid selected from the group consisting of: a) a first nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11 ; b) a second nucleotide sequence of a nucleic acid that selectively hybridizes to a nucleic acid with a nucleotide sequence that is fully complementary to the first nucleotide sequence; c) a third nucleotide sequence that is at least 70% identical to the first nucleotide sequence; and d) a fourth nucleotide sequence of a nucleic acid that encodes the polypeptide encoded by any of the first, second, or third nucleotide sequences.
  • the recombinant expression vector comprises the nucleotide sequence of SEQ ID NO: 2. In another embodiment the recombinant expression vector comprises the nucleotide sequence of SEQ ID NO: 3. In another embodiment the recombinant expression vector comprises the nucleotide sequence of SEQ ID NO: 4. In another embodiment, the recombinant expression vector comprises the nucleotide sequence of SEQ ID NO: 6. In another preferred embodiment, the recombinant expression vector comprises the nucleotide sequence of SEQ ID NO: 7. In another preferred embodiment, the recombinant expression vector comprises the nucleotide sequence of SEQ ID NO: 8. In another preferred embodiment, the recombinant expression vector comprises the nucleotide sequence of SEQ ID NO: 9. In another preferred embodiment, the recombinant expression vector comprises the nucleotide sequence of SEQ ID NO: 10. In another preferred embodiment, the recombinant expression vector comprises the nucleotide sequence of SEQ ID NO: 11.
  • This embodiment of the present invention is a recombinant vector wherein at least one isolated nucleic acid molecule of the present invention is inserted into a vector capable of delivering the nucleic acid molecule into a host cell.
  • a vector contains heterologous nucleic acid sequences (i.e., nucleic acid sequences that are not naturally found adjacent to nucleic acid molecules of the present invention) and are preferably 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, producing, and/or otherwise manipulating of nucleic acids of the Bartonella henselae virB operon 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.
  • an expression vector is a DNA or RNA vector that can transform a host cell and effect expression of a specified nucleic acid molecule.
  • the expression vector is also capable of replicating within the host cell.
  • 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/or plant cells.
  • Preferred expression vectors of the present invention can direct gene expression in bacterial, yeast, insect, and mammalian cells.
  • 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.
  • 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, and/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 pL and lambda pR and fusions that include such promoters), bacteriophage T7, T7lac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoter, antibiotic resistance gene, baculovirus, Heliothis zea 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, Rou
  • transcription control sequences include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).
  • Transcription control sequences of the present invention can also include naturally occurring transcription control sequences naturally associated with Bartonella, such as ⁇ . henselae and/or B. 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 polypeptide of the Bartonella henselae virB operon 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.
  • 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.
  • fusion segment nucleic acids Suitable fusion segments encoded by fusion segment nucleic acids are disclosed herein.
  • 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.
  • the current invention is a prokaryotic or eukaryotic host cell transformed with a nucleic acid comprising a nucleic acid sequence selected from the group consisting of: a) a first nucleotide sequence selected from the group consisting of
  • SEQ ID NO: 1 SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; b) a second nucleotide sequence of a nucleic acid that selectively hybridizes to a nucleic acid with a nucleotide sequence that is fully complementary to the first nucleotide sequence; c) a third nucleotide sequence that is at least 70% identical to the first nucleotide sequence; and d) a fourth nucleotide sequence of a nucleic acid that encodes the polypeptide encoded by the first, second, or third nucleotide sequence.
  • a transformed host cell according to this embodiment of the current invention may be transformed with one or more nucleic acids. Transformation of a nucleic acid molecule, such as a recombinant expression vector, 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, infection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. Transformation may be stable or transient.
  • 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 immortalized 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.
  • Suitable host cells 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 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 polypeptides encoded by the Bartonella virB operon of the present invention or can be capable of producing such polypeptides 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 polypeptide of the present invention, and include bacterial, fungal (including yeast), parasite (including helminth, protozoa, and ectoparasite), insect, animal, and plant cells.
  • Preferred host cells include bacterial, mycobacterial, yeast, insect, and mammalian cells.
  • More preferred host cells include Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces, Spodoptera, Mycobacteha, Trichoplusia, 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 Escherichia coli, including E.
  • coli K-12 derivatives Salmonella typhi; Salmonella typhimurium, including attenuated strains such as UK-1 3987 and SR-11 4072; Spodoptera frugiperda; Trichoplusia ni; BHK cells; MDCK cells; CRFK cells; CV-1 cells; COS cells; Vero cells; and non-tumorigenic 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, murine, or chicken embryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovary cells, mouse NIH/3T3 cells, LMTK31 cells, and/or HeLa cells.
  • 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.
  • 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 henselae virB operon nucleic acid molecules encoding one or more polypeptides 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 nucleic acid 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.
  • the current invention is an isolated polypeptide encoded by a nucleic acid comprising a sequence selected from the group consisting of: a) a first nucleotide sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11 ; b) a second nucleotide sequence of a nucleic acid that selectively hybridizes to a nucleic acid with a nucleotide sequence that is fully complementary to the first nucleotide sequence; c) a third nucleotide sequence that is at least 70% identical to the first nucleotide sequence; and d) a fourth nucleotide sequence of a nucleic acid that encodes the polypeptide encoded by the first, second, or third nucleotide sequences
  • the isolated polypeptide is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 2. In another embodiment the isolated polypeptide is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 3. In another embodiment the isolated polypeptide is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 4. In another embodiment, the isolated polypeptide is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 6. In another preferred embodiment, the isolated polypeptide is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 7.
  • the isolated polypeptide is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 8. In another preferred embodiment, the isolated polypeptide is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 9. In another preferred embodiment, the isolated polypeptide is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 10. In another preferred embodiment, the isolated polypeptide is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 11.
  • the minimum size of a Bartonella virB operon nucleic acid molecule used to encode a polypeptide of the present invention is from about 12 to about 18 nucleotides in length.
  • the minimal size of a polypeptide of the present invention is from about 4 to about 6 amino acids in length.
  • a nucleic acid molecule of the present invention can include a portion of a virB operon- related sequence, the entire virB operon, as well as additional sequences.
  • the preferred size of a polypeptide encoded by a nucleic acid molecule of the present invention depends on whether a full-length, fusion, multivalent, or functional portion of such a polypeptide is desired.
  • polypeptides of the present invention encoded by the virB operon of Bartonella henselae is a fusion protein that includes a domain of a polypeptide encoded by the Bartonella henselae virB operon 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 polypeptide encoded by the Bartonella henselae virB operon; and/or assist in purification of a polypeptide encoded by the Bartonella henselae virB operon (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 or increased immunogenicity on a protein, and/or simplifies purification of a protein). Fusion segments can be joined to amino and/or carboxyl termini of the domain containing the polypeptide encoded by the Bartonella henselae virB operon and can be susceptible to cleavage in order to enable straight-forward recovery of a polypeptide encoded by the Bartonella henselae virB operon.
  • 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 domain containing a polypeptide encoded by the Bartonella henselae virB operon.
  • 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 $-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).
  • 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
  • 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.
  • Polypeptides encoded by the Bartonella henselae virB operon can be the result of natural allelic variation or natural mutation.
  • Polypeptides encoded by the Bartonella henselae virB operon of the present invention can also be produced using techniques known in the art including, but not limited to, direct modifications to the polypeptides or modifications to the nucleic acid molecule encoding the polypeptide using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis.
  • a preferred polypeptide encoded by the Bartonella henselae virB operon 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 B. henselae or ⁇ . quintana infection. In one embodiment, such a polypeptide protects an animal from cat scratch disease.
  • the ability of a polypeptide encoded by the Bartonella henselae virB operon of the present invention to protect an animal from Bartonella infection refers to the ability of that polypeptide to, for example, treat, ameliorate, and/or prevent Bartonella infection, and preferably ⁇ . henselae or B. quintana infection.
  • polypeptides encoded by the Bartonella henselae virB operon and isolated nucleic acids related to the Bartonella henselae virB operon refer to polypeptides and nucleic acid molecules, respectively, that can be isolated from an organism or prepared recombinantly or synthetically.
  • cat scratch disease refers to the group of diseases most normally caused by the bacterium B. henselae. Some animals (e.g., the domestic cat) do not get a disease perse when infected with B. henselae.
  • ⁇ The term " ⁇ .
  • henselae infection is used herein to denote infection with this bacterium, either with or without the subsequent development of detectable disease.
  • the term "to protect” includes, for example, to prevent or to treat (e.g., reduce or cure) B. henselae infection in the subject animal.
  • a therapeutic composition of the present invention can be a prophylactic vaccine or a treatment for animals already infected with the organism.
  • An isolated polypeptide encoded by the Bartonella henselae virB operon of the present invention includes a B. 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 polypeptide.
  • the present invention also includes mimetopes of polypeptides encoded by the Bartonella henselae virB operon.
  • a mimetope of a polypeptide encoded by the Bartonella henselae virB operon of the present invention refers to any compound that is able to mimic the activity of such a polypeptide encoded by the Bartonella henselae virB operon, often because the mimetope has a structure that mimics the particular polypeptide encoded by the Bartonella henselae virB operon.
  • 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 molecules, such as oligonucleotides, peptides or other organic molecules, and screening such samples by affinity chromatography techniques using the corresponding binding partner.
  • Polypeptides encoded by the Bartonella henselae virB operon of the present invention can be produced in a variety of ways, including methods known in the art involving production and recovery of natural proteins, methods involving production and recovery of recombinant proteins, and chemical synthesis of the polypeptides.
  • One method of producing the polypeptides encoded by the Bartonella henselae virB operon of the present invention is a method for producing polypeptides in a host cell, described below.
  • polypeptides of the present invention can be produced from natural sources.
  • Bartonella henselae 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.
  • Bartonella henselae can be grown upon a eukaryotic cell monolayer of, for example, 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 henselae 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 henselae can also be grown in bacteriological broth suspension culture, for example, in supplemented Brucella broth. Bartonella henselae 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.
  • One preferred embodiment of the present invention includes isolated polypeptides with an amino acid sequence listed as SEQ ID NO: 14.
  • the expected molecular weight of this protein is 89.5 kDa. This size closely resembles the size (83 kDa) of an immunodominant antigen of ⁇ . henselae, the Bartonella Bh83 protein, that has been shown to be reactive with sera from patients diagnosed with cat scratch disease (CSD) (McGill.et al, "Characterization of human immunoglobulin (Ig) isotype and IgG subclass response to Bartonella henselae infection". Infection and Immunity, 66: 5915 (1998). Bartonella Bh83 proteins are immunoreactive with serum from a patient having antibodies against B. henselae. A Bartonella Bh83 protein is a ⁇ .
  • 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 B. henselae Bh83 protein of 83 kDa.
  • Such a protein while immunoreactive with serum from patients having antibodies against ⁇ . henselae, is not immunoreactive with serum against an organism selected from the group consisting of: Rickettsia rickettsii, Chlamydia spp., Treponema pallidum, Orientia tsutsugamushi,
  • the current invention is a method for producing a Bartonella henselae virB operon polypeptide from a prokaryotic or eukaryotic host cell comprising the steps of: a) introducing a Bartonella henselae virB operon nucleic acid which is capable of expressing a polypeptide in the prokaryotic or eukaryotic host cell into a vector, thereby producing a Bartonella henselae virB operon polypeptide expression vector; b) introducing the Bartonella henselae virB operon polypeptide expression vector into the prokaryotic or eukaryotic host cell to produce an engineered host cell; c) maintaining the engineered host cell under conditions suitable for the expression of the polypeptide by the engineered host cell; and d) recovering the polypeptide produced by the engineered host cell.
  • the polypeptide is the polypeptide encoded by
  • polypeptide is the polypeptide encoded by SEQ ID NO: 12. In another embodiment, the polypeptide is the polypeptide encoded by SEQ ID NO: 13. In another embodiment, the polypeptide is the polypeptide encoded by SEQ ID NO: 14. In another embodiment, the polypeptide is the polypeptide encoded by SEQ ID NO: 16. In another embodiment, the polypeptide is the polypeptide encoded by SEQ ID NO: 17. In another embodiment, the polypeptide is the polypeptide encoded by SEQ ID NO: 18. In another embodiment, the polypeptide is the polypeptide encoded by SEQ ID NO: 19. In another embodiment, polypeptide is the polypeptide encoded by SEQ ID NO: 20. In another embodiment, the polypeptide is the polypeptide encoded by SEQ ID NO: 21.
  • a preferred cell to culture is a host cell engineered to possess nucleic acids of the Bartonella henselae virB operon.
  • 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 polypeptide encoded by the Bartonella henselae virB operon 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.
  • resultant polypeptides 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.
  • polypeptides 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. Polypeptides of the present invention are preferably retrieved in "substantially pure" form.
  • 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 current invention is a method of detecting current or previous infection with Bartonella henselae in a subject by detecting the presence of an antibody that specifically binds an isolated polypeptide of the B. henselae virB operon or immunogenic fragment thereof, said method comprising: a) contacting an antibody-containing fluid or tissue sample from the subject with an amount of the isolated polypeptide or immunogenic fragment thereof which binds to the antibody, the isolated polypeptide or immunogenic fragment encoded by a nucleic acid related to the virB operon of ⁇ .
  • henselae comprising a nucleotide sequence selected from the group consisting of: i) a first nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11 ; ii) a second nucleotide sequence of a nucleic acid that selectively hybridizes to a nucleic acid comprising a nucleotide sequence that is complimentary to the first nucleotide sequence; iii) a third nucleotide sequence that is at least 70% identical to the first nucleotide sequence of a); and iv) a fourth nucleotide sequence of a nucleic acid that encodes the polypeptide encoded by the first, second, or third nucleotide sequence; b) detecting the binding of the isolated polypeptide or the immunogenic fragment to the antibody
  • the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 14.
  • B. quintana a preferred Bartonella species to detect using methods of the present invention.
  • the B. henselae antigen can be bound to a substrate and contacted by a fluid sample such as blood, plasma, or serum. This sample can be taken directly from the patient or can be partially purified. Where the fluid is taken directly from the patient, it can comprise any body fluid which would contain B. henselae, for example, blood, plasma, and serum. Other possible examples of body fluids include urine, sputum, mucus, and the like.
  • antibody which specifically binds with polypeptides encoded by the Bartonella henselae virB operon indicates the presence of infection by B. henselae.
  • the term "specifically binds” denotes that an antibody or other ligand does not cross-react, or bind, substantially with any antigen other than the one specified, in this case, a polypeptide encoded by the Bartonella henselae virB operon.
  • Antibodies specific for ⁇ . henselae antigen will specifically bind with the bound B. henselae antigen. Thereafter, a secondary antibody bound to, or labeled with, a detectable moiety can be added to enhance the detection of the primary antibody.
  • the secondary antibody will be selected for its ability to bind with multiple sites on the primary antibody. Thus, for example, several molecules of the secondary antibody can bind with each primary antibody, making the primary antibody more detectable.
  • a detectable moiety will preferably allow visual detection of a precipitate or a color change, visual deletion by microscopy, or automated detection by spectrometry or radiometric measurement, or the like.
  • detectable moieties include fluorescein and rhodamine (for fluorescence microscopy), horseradish peroxidase (for light microscopy, electron microscopy, or biochemical detection), biotin/strepavidin (for light or electron microscopy), and alkaline phosphatase (for biochemical detection by color change).
  • the detection method and detectable moiety used can be selected by standard criteria applied to such selections as described in Harlow and Lane, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1988)).
  • Detecting methods such as immunofluorescence assays (IFA) and enzyme linked immunosorbent assays (ELISA) can be readily adapted to accomplish the detection of both B. henselae antigen and antibodies which specifically bind therewith.
  • An example of an ELISA method effective for the diagnosis of B. henselae infection based on the detection of human IgG-type antibodies can be performed as follows: (1) bind at least one B. henselae polypeptide of the present invention to a substrate; (2) contact the bound polypeptide with a serum sample from a subject which possibly contains IgG- type antibodies reactive with the B.
  • henselae polypeptide to form a polypeptide/antibody complex; (3) remove serum components that are not part of the polypeptide/antibody complex; (4) contact the polypeptide/antibody complex with an anti-human IgG antibody (secondary antibody) bound to a detectable moiety (e.g., horseradish peroxidase enzyme or alkaline phosphatase enzyme); (5) rinse away unbound secondary antibody; (6) contact the polypeptide/antibody complex with a substrate for the enzyme which changes color upon an enzyme-catalyzed reaction to form a product; and (7) observe color change in the substrate after the enzyme-catalyzed reaction.
  • an anti-human IgG antibody secondary antibody bound to a detectable moiety
  • a detectable moiety e.g., horseradish peroxidase enzyme or alkaline phosphatase enzyme
  • a modification of the above ELISA effective for diagnosis of B. henselae infection angiomatosis based on the detection of human IgM antibodies can be as follows: (1) bind an anti-human IgM antibody capable of reacting with a human IgM antibody to a substrate (antibody capture); (2) contact the bound antibody with a serum sample from a subject; (3) contact the above with B. henselae antigen; (4) contact the above with a rabbit anti- ⁇ .
  • henselae antibody (5) contact the above with an anti-rabbit antibody bound to a detectable moiety (e.g., horseradish peroxidase enzyme); (6) contact the above with substrate for the enzyme; (7) contact the above with a color reagent; (8) observe a color change in the presence of an IgM antibody specifically reactive with ⁇ . henselae antigen.
  • a detectable moiety e.g., horseradish peroxidase enzyme
  • (6) contact the above with substrate for the enzyme (7) contact the above with a color reagent; (8) observe a color change in the presence of an IgM antibody specifically reactive with ⁇ . henselae antigen.
  • IgM capture ELISA flat-bottomed 96-well polystyrene plates can be coated with goat anti-human IgM antibody, followed by serial two-fold dilutions of sera including 5 negative controls, B. henselae or negative control antigens, B.
  • henselae hyperimmune rabbit antisera, and goat anti-rabbit conjugated to horseradish peroxidase are incubated for 1 hour at 37° C, and then washed 3 times with 0.1% Tween 20 in PBS. The final step is incubation in the substrate ABTS in a suitable buffer after a rinse in PBS. After this final step, absorbance is read at a wavelength of 410 nm using, for example, an automated plate reader. Dilutions of sera are considered positive when the difference in absorbance between that serum specimen when tested with B. henselae antigen and the negative control antigen exceeds the mean plus 3 standard deviations of the 5 negative control sera tested with both B. henselae and negative control antigens.
  • immunoblot analysis can be readily adapted to detect the presence of B. henselae antigen and antibodies which specifically bind therewith.
  • An immunoblot analysis effective for the detection of a polypeptide encoded by the ⁇ . henselae virB operon can, for example, be as follows: (1) grow recombinant vectors containing B.
  • henselae virB operon nucleic acids in an appropriate host for expression of the recombinant nucleic acid; (2) induce the host to express the recombinant nucleic acid; (3) solubilize the host to release the polypeptide expressed by the vector; (4) electrophorese the released polypeptides on sodium dodecyl sulfate- polyacrylamide gels; (5) transfer the proteins to nitrocellulose membranes; (6) incubate the nitrocellulose membranes with serum from patients potentially infected with ⁇ . henselae; and (7) detect the bound antigen or antibody by reacting the filters with goat anti-human IgG conjugated with horseradish peroxidase.
  • Another method of the present invention that can be useful in the detection of ⁇ . henselae infection utilizes monoclonal antibodies for detection of antibodies which specifically bind with polypeptides of the B. henselae virB operon. Briefly, sera from the subject is incubated with B. henselae antigen bound to a substrate (e.g., an ELISA 96-well plate). After excess sera is thoroughly washed away, a labeled (enzyme-linked, fluorescent, radioactive, etc.) monoclonal antibody is incubated with the previously reacted antigen- serum antibody complex. The amount of inhibition of monoclonal antibody binding is measured relative to a control. This method is very specific for a particular species since it is based on monoclonal antibody binding specificity.
  • a substrate e.g., an ELISA 96-well plate
  • the method comprises contacting a fluid or tissue sample from the subject with an amount of a purified ligand (e.g., antibodies or antibody fragments) that specifically bind polypeptides encoded by the ligand (e.g., antibodies or antibody fragments) that specifically bind polypeptides encoded by the ligand (e.g., antibodies or antibody fragments) that specifically bind polypeptides encoded by the ligand (e.g., antibodies or antibody fragments) that specifically bind polypeptides encoded by the
  • Bartonella henselae virB operon and detecting the reaction of the ligand with polypeptides encoded by the Bartonella henselae virB operon.
  • the term "ligand" includes an intact antibody, a fragment of an antibody, or another reagent that binds nonrandomly with the antigen.
  • infection of ⁇ . henselae can be determined by detecting the presence of a nucleic acid related to the virB operon of B. henselae.
  • current or previous Bartonella henselae infection of a subject is detected using the presence of nucleic acids of the Bartonella henselae virB operon.
  • This method comprises: a) contacting a sample from the subject with an amount of a detection nucleic acid that is at least 70% identical to a nucleotide sequence of at least 10 continuous nucleotides of the Bartonella henselae virB operon, wherein the continuous nucleotide sequence is not from SEQ ID NO: 22; b) detecting hybridization of the detection nucleic acid to a target nucleic acid found in the sample; and c) correlating the presence of the target nucleic acid in the sample with infection of Bartonella henselae in the subject.
  • the nucleic acid is at least 35 nucleotides in length. In another embodiment, the nucleic acid is at least 50 nucle
  • the nucleic acid can be detected by the presence of an amplification product following polymerase chain reaction (PCR), or other routine amplification method, using primers specific for the Bartonella henselae virB operon.
  • PCR polymerase chain reaction
  • the nucleic acid can be detected by probing nonspecific amplification products of PCR with a probe specific for nucleic acids related to the Bartonella henselae virB operon.
  • a probe specific for nucleic acids related to the Bartonella henselae virB operon can be used in an in situ hybridization protocol to detect the presence of a nucleic acid sequence specific for the organism.
  • lymph node biopsy tissue from a patient suspected of having a disease associated with Bartonella infection such as cat scratch disease ("CSD"
  • CSD cat scratch disease
  • B. quintana is genetically similar to ⁇ . henselae
  • the instant invention also provides a method of detecting past or current infection of B. quintana.
  • the method of detecting infection of B. quintana can be accomplished using similar protocols to those taught above for the detection of ⁇ . henselae. These protocols include those described above related to detecting polypeptides as well as nucleic acids.
  • This method can be used to detect bacillary angiomatosis ("BA") since B. quintana is associated with BA (Koehler, J. E., et al. Isolation of Rochalimaea species from cutaneous and osseous lesions of bacillary angiomatosis, N. Engl. J. Med. 327:1625 (1992)).
  • Diagnostic kits for detecting Bartonella henselae In another preferred embodiment, the current invention is a diagnostic kit either for detecting nucleic acids related to the virB operon of ⁇ . henselae, or for detecting antibodies against polypeptides encoded by the virB operon of B. henselae.
  • Such a kit comprises: a) an isolated nucleic acid related to the Bartonella henselae virB operon, said isolated nucleic acid comprising a nucleotide sequence of at least 50 nucleotides in length selected from the group consisting of: i) a first nucleotide sequence of SEQ ID NO: 1 , wherein the first nucleotide sequence is not from SEQ ID NO: 22; ii) a second nucleotide sequence that is complimentary to the first nucleotide sequence; iii) a third nucleotide sequence of a nucleic acid that selectively hybridizes to the second nucleotide sequence; and iv) a fourth nucleotide sequence that is at least 70% identical to the first nucleotide sequence; and b) a reagent for detecting binding of the nucleic acid to nucleic acids in a sample.
  • the reagent for detecting binding of the nucleic acid is a reagent for performing a hybridization or amplification method (e.g., Southern hybridization, dot blot hybridization, PCR, SDA, and the like) such as a second nucleic acid that can complement the nucleic acid of the invention as a primer in an amplification reaction, or an enzyme for amplifying a target nucleic acid upon binding of a primer.
  • the kit may further include other components and reagents for performing a hybridization or amplification method, as well as instructions for carrying out the analysis.
  • a hybridization solution See Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL (2d ed.
  • the current invention is a diagnostic kit comprising: a) an isolated polypeptide or an immunogenic fragment thereof capable of being bound by an antibody of subjects exposed to Bartonella henselae, said isolated polypeptide encoded by a nucleic acid comprising a nucleotide sequence selected from the group consisting of: i) a first nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; ii) a second nucleotide sequence of a nucleic acid that selectively hybridize to a nucleic acid comprising a nucleotide sequence that is complimentary to the first nucleotide sequence; iii) a third nucleotide sequence that is at least 70% identical to the first nucleot
  • kits for any of the embodiments described above are typically packaged together in a common container, including written instructions for performing selected specific embodiments of the methods disclosed herein.
  • Components for detection methods, as described hereinabove, may optionally be included in the kit.
  • Such components could include, for example, a second probe, and/or reagents and means for performing label detection (e.g., radiolabel, enzyme substrates, antibodies, etc., and the like).
  • label detection e.g., radiolabel, enzyme substrates, antibodies, etc., and the like.
  • the current invention is a therapeutic composition against Bartonella henselae infection, said therapeutic composition comprising an effective amount of a Bartonella henselae virB operon-related compound and a pharmaceutically acceptable carrier.
  • the Bartonella henselae virB operon-related compound is a nucleic acid comprising a nucleotide sequence selected from the group consisting of: i) a first nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11 ; ii) a second nucleotide sequence of a nucleic acid that selectively hybridizes to a nucleic acid comprising a nucleotide sequence that is complimentary to the first nucleotide sequence; iii) a third nucleotide sequence
  • a therapeutic composition of the present invention when administered to an animal in an effective manner, is capable of protecting that animal from Bartonella infection, and preferably from B. henselae infection.
  • Bartonella henselae wr ⁇ -related compounds of the present invention include at least one of the following protective compounds: an isolated polypeptide encoded by the Bartonella henselae virB operon or a mimetope thereof, an isolated Bartonella henselae virB operon nucleic acid molecule, an isolated antibody that selectively binds to an isolated polypeptide encoded by the Bartonella henselae virB operon, an inhibitor of function of an isolated polypeptide encoded by the Bartonella henselae virB identified by its ability to bind to an isolated polypeptide encoded by the Bartonella henselae virB operon, and mixtures thereof (i.e., combination of at least two of the compounds whether they are combined in a single mixture or not).
  • 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 B. henselae infection.
  • Examples of polypeptides, nucleic acid molecules, antibodies, and inhibitors of the present invention are disclosed herein.
  • the present invention also includes a therapeutic composition comprising at least one Bartonella henselae virB operon-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 ⁇ . henselae 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 ⁇ . henselae 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.
  • compositions of the present invention can be formulated in an excipient that the animal to be treated can tolerate.
  • 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, or triglycerides 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.
  • buffers examples include phosphate buffer, bicarbonate buffer, and Tris buffer; 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.
  • the excipient in a non-liquid formulation, can comprise dextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration.
  • 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), interleukin-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
  • 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, liposomes, bacteria, viruses, other cells, oils, esters, and glycols.
  • a controlled release formulation that is capable of slowly releasing a composition of the present invention into an animal.
  • 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, liposomes, 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 and/or 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 rate (preferably at an essentially constant rate) sufficient to attain therapeutic dose levels of the composition to protect an animal from Bartonella infection and preferably from B. henselae 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.
  • 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 including B. quintana infection, and preferably B. henselae infection.
  • B. quintana infection preferably B. henselae infection.
  • polypeptides, 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.
  • a preferred single dose of a protein, polypeptide, or antibody therapeutic composition is from about 1 microgram ( ⁇ g) to about 10 milligrams (mg) of the 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, or just before, 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 ⁇ g 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.
  • the therapeutic composition is a nucleic acid molecule of the present invention related to the Bartonella henselae virB operon.
  • the nucleic acid can be administered to an animal in a fashion to enable expression of that nucleic acid molecule to form a protective protein or protective RNA (e.g., antisense RNA, ribozyme, triple helix forms, or RNA drug) in the animal.
  • a protective protein or protective RNA e.g., antisense RNA, ribozyme, triple helix forms, or RNA drug
  • 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., naked DNA or RNA molecules, such as is taught, for example in Wolff et al., Science 247, 1465-1468 (1990)) 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 naked nucleic acid as a genetic vaccine e.g., naked DNA or RNA molecules, such as is taught, for example in Wolff et al., Science 247, 1465-1468 (1990)
  • a nucleic acid molecule packaged as a recombinant virus vaccine or as a recombinant cell vaccine i.e., the nu
  • a genetic (i.e., naked nucleic acid) vaccine of the present invention includes a nucleic acid molecule related to the B. henselae virB operon of the present invention and preferably includes a recombinant molecule of the present invention that preferably includes nucleic acid sequences, such as transcription control sequences, that direct expression of the nucleic acid molecule related to the B. henselae virB operon.
  • 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. Those based on alphaviruses (such as Sindbis or Semliki forest virus), species-specific herpesviruses, and poxviruses are particularly preferred.
  • transcription control sequence can be used, including those 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 ⁇ g, 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, injection, drops, aerosols, and/or topical. 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).
  • aqueous excipient e.g., phosphate buffered saline
  • carrier e.g., lipid-based vehicles
  • the efficacy of a therapeutic composition of the present invention to protect an animal from Bartonella infection including B. quintana infection, and preferably from ⁇ . henselae 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, preferably a ⁇ . henselae microorganism, to determine whether the treated animal is resistant to infection or disease from that microorganism.
  • therapeutic compositions can be tested in animal models such as mice. Such techniques are known to those skilled in the art.
  • One therapeutic composition of the present invention includes an inhibitor of the function of a polypeptide encoded by the B. henselae virB operon (i.e., a compound capable of substantially interfering with the function of a polypeptide encoded by the B. henselae virB operon).
  • 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 polypeptide encoded by the Bartonella henselae virB operon, to protect the animal from the disease.
  • an antibody of the present invention is administered to an animal in an effective manner and amount such that upon interaction of that antibody with a native polypeptide of the Bartonella henselae virB operon, the animal is protected 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 a polypeptide of the Bartonella henselae virB operon.
  • An inhibitor of function of a polypeptide encoded by the B. henselae virB operon can be identified using a polypeptide encoded by the ⁇ . henselae virB operon of the present invention.
  • a preferred method to identify a compound capable of inhibiting activity of a polypeptide encoded by the B. henselae virB operon includes contacting an isolated polypeptide encoded by the ⁇ . henselae virB operon with a putative inhibitory compound under conditions in which, in the absence of said compound, said polypeptide has activity of a polypeptide encoded by the B. henselae virB operon; and determining if said putative inhibitory compound inhibits said activity.
  • the present invention comprises a vaccine that comprises a nucleic acid molecule of the present invention, that when administered to an animal, is capable of protecting that animal from infection with a Bartonella bacterium, and preferably from infection with ⁇ . henselae or ⁇ . quintana which can lead to cat scratch disease, at least in certain animals known to those skilled in the art.
  • 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.
  • the vaccine comprises a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 4.
  • the current invention is a vaccine against Bartonella henselae infection, wherein the vaccine comprises an immunogenic amount of an isolated polypeptide and a pharmaceutically acceptable carrier, the isolated polypeptide encoded by a nucleic acid comprising a nucleotide sequence selected from the group consisting of: i) a first nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11 ; ii) a second nucleotide sequence of a nucleic acid that selectively hybridizes to a nucleic acid comprising a nucleotide sequence complimentary to the first nucleotide sequence; iii) a third nucleotide sequence that is at least 70% identical to the first nucleotide sequence; and iv) a fourth nucleotide sequence of
  • the vaccine can be used in a method of preventing a disease resulting from infection with ⁇ . henselae such as cat scratch disease in a subject by administering the vaccine to the subject. Other disease syndromes are associated with B. henselae infection can also be prevented by use of the vaccines of this invention.
  • the prevention methods will work when the subject is a human, or a nonhuman animal (e.g., a cat).
  • the pharmaceutically acceptable carrier in the vaccine of the instant invention can comprise saline or other suitable carriers.
  • An adjuvant can also be a part of the carrier of the vaccine, in which case it can be selected by standard criteria based on the particular R. henselae antigen used, the mode of administration, and the subject. Methods of administration can be oral, subiingual, or injection, depending on the particular vaccine used and the subject to whom it is administered.
  • the vaccine can be used as a prophylactic or a therapeutic.
  • subjects with the disease can be treated utilizing the vaccine.
  • the spread of disease between animals and humans can be prevented, or at least substantially reduced.
  • cats or dogs can be immunized, thereby significantly reducing the risk to humans.
  • Immunogenic amounts of the isolated polypeptides of vaccines of the current invention can be determined using standard procedures. Briefly, various concentrations of the polypeptide are prepared and administered to test animals. The immunological response (i.e., the production of antibodies) of an animal to each concentration is determined.
  • the invention provides methods of preventing or treating a ⁇ . henselae infection and the associated disease by administering the vaccine to a subject.
  • 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.
  • 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 ar ⁇ ino acids.
  • a polypeptide of a vaccine of the present invention includes at least one additional protein segment that is capable of protecting an animal from one or more diseases.
  • 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.
  • multivalent protective compounds include, but are not limited to, a polypeptide encoded by a nucleic acid related to the Bartonella henselae virB operon 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, Capno
  • the vaccine is a recombinant virus vaccine.
  • 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.
  • 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, picomaviruses, and retroviruses.
  • Preferred recombinant virus vaccines are those based on alphaviruses (such as Sindbis virus), raccoon poxviruses, species-specific herpesviruses, and species-specific poxviruses.
  • alphaviruses such as Sindbis virus
  • raccoon poxviruses species-specific herpesviruses
  • species-specific poxviruses 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.
  • 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, including B.
  • a recombinant virus vaccine comprising a nucleic acid molecule related to the B. henselae virB operon 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, including B. quintana infection, and preferably from B. henselae infection.
  • a preferred single dose of a recombinant virus vaccine of the present invention is from about 1 x 10 4 to about 1 x 10 8 virus plaque forming units (pfu) per kilogram body weight of the animal.
  • 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 10 8 to about 10 12 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.
  • Antibodies against polypeptides encoded by the virB operon also includes isolated (i.e., removed from their natural milieu) antibodies that selectively bind to a polypeptide encoded by the Bartonella henselae virB operon of the present invention or a mimetope thereof.
  • the terms "selectively binds to” or “is immunoreactive with” a polypeptide 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, and the like.
  • 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.
  • antibodies of the present invention are produced recombinantly using techniques as heretofore disclosed to produce polypeptides 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.
  • antibodies of the present invention 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 B. henselae or B. quintana infection, and/or (c) as tools to screen expression libraries and/or to recover desired polypeptides of the present invention from a mixture of proteins and other contaminants.
  • antibodies of the present invention can be used to target cytotoxic agents to Bartonella in order to 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.
  • the following examples describe and illustrate the methods and compositions of the invention. These examples are intended to be merely illustrative of the present invention, and not limiting thereof in either scope or spirit. Those of skill in the art will readily understand that variations of the materials used in, and the conditions and processes of, the procedures described in these examples can be used.
  • EXAMPLE 1 Cloning of the 17kDa fi. henselae antigen. Isolation and characterization was performed of nucleic acids encoding antigens of B. henselae. B. henselae (Houston-1 , ATCC # 49882 ) was cultivated on heart infusion agar plates supplemented with 5% rabbit blood (BBL, Cockeysville, Md.). Plates streaked with B. henselae were incubated for 3 to 5 days at 32°C in the presence of 5% C0 2 . Bacteria were harvested in a laminar flow hood by gently scraping colonies off the agar surface in brain heart infusion (BHI) broth media. The cells were then collected via centrifugation and suspended in phosphate-buffered saline solution (PBS) or TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA).
  • PBS phosphate-buffered saline solution
  • TE buffer 10
  • E.co// strains XL-1B/we and SOLR (Stratagene, La Jolla, CA) were grown at 37°C on Luria plates supplemented with tetracycline (12.5 ⁇ g/ml.) and kanamycin (50 ⁇ g/ml.), respectively.
  • E.co// strains harboring recombinant vectors were grown on Luria plates supplemented with ampicillin (50 ⁇ g/ml.).
  • Genomic DNA from B. henselae was extracted by a protocol that has been described earlier (Anderson et al., 1994). Genomic DNA was extracted from suspensions of bacteria by digestion with proteinase K (100 ng per ml) in the presence of 1% SDS for 90 min at 55 °C. The resulting lysate was extracted two to three times with a 50:50 mixture of chloroform-isoamyl alcohol/ phenol. The DNA was then precipitated from the aqueous supernatant by adding 0.1 volume of 3M sodium acetate and 2.5 volumes of ethanol.
  • Plasmid DNA was isolated by using the QIAGEN mini-prep protocol.
  • B. henselae genomic DNA was partially digested with the restriction endonuclease, 7sp509l (New England Biolabs, Beverly, MA) (65 °C for 30 min.). Fragments ranging in size from 2 to 8 kb were ligated to EcoRI- digested, alkaline phosphatase-treated lambda Zapll vector arms, using the lambda Zapll cloning system (Stratagene).
  • the ligation products were packaged in vitro using the Gigapackll Gold packaging system (Stratagene). Packaged lambda phage was then used to infect the E.coli strain, XL-1 ⁇ /ue. The titer of the resulting library was estimated to be approximately 4 X 10 9 plaque-forming units (pfu) per ml.
  • Colony-forming units of harvested Bartonella cultures were titered on blood agar plates prior to being frozen and stored at -70°C until use.
  • BALB/c mice (4-6 weeks old) were obtained from Harlan Sprague- Dawley (Indianapolis, Ind.). Mice were anesthetized with methoxyflurane (Metofane; Pitman-Moore Inc., Mundelein, II) and administered live B. henselae at doses of 3 x 10 6 CFU/mouse subcutaneously. A second subcutaneous inoculation was given 14 days following the first inoculation. Blood was drawn from individual mice on day 28, serum collected and pooled. Sera were tested for recognition of B. henselae cellular antigen lysates by Western blot analysis. Sera were then frozen at -70 °C until they were used for screening the genomic library.
  • the ⁇ . henselae lambda Zapll library was screened using mice anti- Bartonella antiserum generated as described in the preceding section. The sera were diluted 1 :200 in 5% BLOTTO for immunoscreening. The immunoscreening was done as described previously (Padmalayam et al., 1997). The B. henselae library was plated on 150 mm Petri plates at a density of approximately 50,000 plaques per plate. Plates were incubated at 42 °C for 3.5 hours until the plaques were just visible.
  • Nitrocellulose membrane circles (137 mm) that were treated with 10 mM IPTG ( isopropyl- ⁇ - D-thiogalactopyranoside) were placed on the surface of the agar and the orientation was marked by using an asymmetric pattern. The plates were then incubated at 37°C for 3 hours. After incubation, the membranes were removed and washed once in TBST (Tris-Buffered Saline with Tween-20) and stored overnight in 5% BLOTTO at 4°C. The membranes were then incubated in the primary antibody solution consisting of polyclonal mice antisera raised to live B. henselae (diluted 1 :100 in 5% BLOTTO), for 2 hours at room temperature in an orbital shaker.
  • IPTG isopropyl- ⁇ - D-thiogalactopyranoside
  • Membranes were washed with TBST four times (5 min per wash), and incubated in the secondary antibody (anti-human IgG conjugated to horse radish peroxidase (HRP) (diluted 1 :5000 in 5% BLOTTO) for 1 hr at room temperature in an orbital shaker. Membranes were washed four times in TBST (5 min per wash) and once in TBS (Tris- Buffered Saline). The membranes were then treated with peroxidase substrate solution (100 ml-200 ml per membrane) to visualize immunoreactive plaques. The reaction was stopped by rinsing with deionized water and the membranes were dried.
  • HRP horse radish peroxidase
  • Immunoreactive plaques were picked and stored in 1 ml phage dilution buffer containing 1 % chloroform at 4°C overnight to allow the phage to elute into the buffer.
  • the plaques were purified by plating the buffer and immunoscreening according to the protocol described above. Plaque hybridizations were performed using the Genius nonradioactive labeling system (Boehringer Mannheim (now Roche Diagnostics), Indianapolis, IN). The probes used for the plaque hybridization were labeled with digoxygenin-dUTP by using the PCR DIG probe synthesis system
  • the membranes were then sequentially treated with the following solutions in the give order: (1) denaturation solution (0.5 N NaOH, 1.5 M NaCl) for 10 min; (2) neutralization solution (1.0 M Tris-HCl, pH 7.5, 1.5 M NaCl) for 15 min; and (3) 2X SSC (0.3M NaCl, 30 mM sodium citrate, pH 7.0) for 15 min.
  • the transferred DNA was crosslinked to the membrane with UV light and then incubated in 3X wash solution ( 3X SSC, 0.1% SDS) at 68°C for 3 hr to remove cellular debris.
  • the membranes were prehybridized by incubating in DIG EasyHyb solution ( Boehringer Mannheim) at the hybridization temperature with gentle rocking for 2 hr.
  • the membranes were then incubated in the hybridization solution ( DIG EasyHyb solution containing the denatured probe at a concentration of 5-25 ng/ml) at the hybridization temperature for 16 hr with gentle rocking.
  • the membranes were washed twice for 5 min in 2X wash solution (2X SSC, 0.1% SDS) at room temperature with gentle agitation, followed by two 15 min washes in 0.5X wash solution (0.5X SSC, 0.1% SDS) at 68°C.
  • the air-dried membranes were equilibrated in 1X Washing Buffer (Boehringer Mannheim) for 1 min, and incubated in 1X Blocking Buffer (Boehringer Mannheim) for 30-60 min.
  • the membranes were incubated in DIG-labeled alkaline phosphatase solution (diluted 1:5000 in 1X Blocking Buffer) for 30 min at room temperature, and then washed twice with 1X Washing Buffer (Boehringer Mannheim) (15 min per wash). After equilibration in 1X Detection Buffer (Boehringer Mannheim), the membranes were treated with NBT( 75 mg/ml nitroblue tetrazolium salt in dimethylformamide)/BCIP (50 mg/ml 5-bromo-4-chloro-3-indoyl phosphate, toluidium salt in dimethylformamide) solution (diluted in 1X Detection Buffer) and positive plaques were identified and picked. As described in the protocol for immunoscreening, the plugs containing positive plaques were placed in phage dilution buffer containing chloroform. The plaques were purified by plating the plug supernatant and repeating the plaque hybridization as described above.
  • the membrane was blocked with 5% BLOTTO and reacted with individual human sera (1 :200 dilutions) for 2 hours by using a Mini Protean II Multiscreen System (Bio-Rad, Hercules, CA). The membrane was then reacted with a 1 :5000 dilution of horseradish peroxidase-conjugated goat anti- human antibody (Kirkegaard and Perry, Gaithersburg, MD) for 1 hour. The membrane was developed with a TMB membrane substrate developer (Kirkegaard and Perry).
  • DNA sequencing of plasmid inserts was performed using a model 377 automated nucleic acid sequencer (Applied Biosystems, Foster City, CA). DNA and protein analysis was done using the Wisconsin software package of the Genetics Computer Group (GCG, Madison, WI), Lasergene (DNASTAR, Inc., Madison, WI) and MacDNASIS (Hitachi Software Engineering America Ltd., San Bruno, CA).
  • the nucleotide sequence of the 3074 base pair (bp) insert within pBHIM-2 revealed three complete and two partial open reading frames.
  • One of the open reading frames was 483 bp long (SEQ ID NO: 5) and was capable of encoding a protein of 160 amino acids (18 kDa ).
  • This open reading frame was designated as ORF-17.
  • a search through the nucleic acid and protein databases revealed that the predicted amino acid sequence of ORF-17 was identical to that of a 17 kDa antigen that has been previously characterized in B. henselae (Anderson et al., 1995). As noted by Anderson et al., a second in-frame methionine is located 33 bp downstream of the first methionine.
  • This methionine is preceded by a Shine-Dalgarno sequence that is identical to that of E.coli. Also, the size of the open reading frame using the second methionine as start site (16.89 kDa) is closer to the size of the protein observed by SDS-PAGE (17 kDa) (Table 1). The second methionine could be therefore considered as an alternative start site.
  • ORF-17 was subcloned into the expression vector pKK223-3 and the resulting recombinant clone pKKORF-17 was found to express an antigen that migrated around 17 kDa on an SDS-PAGE gel.
  • the protein expressed by pKKORF-17 reacted with sera from patients with cat scratch disease ( Figure 2, lanes 1 to 4), but not with sera that was tested to be negative for Bartonella by IFA ( Figure 2, lanes 7 to 10).
  • the 17 kDa protein does not react with sera from patients with clinical bartonellosis caused by a closely related bacterium, B. bacilliformis ( Figure 2, lanes 5 and 6).
  • EXAMPLE 2 Isolation and Characterization of fi. henselae virB4 Coding Sequences. Isolation and characterization was performed of nucleic acids at the 5' end of ORF-17 (see Example 1). DNA sequencing and analysis as well as plaque hybridization was performed as described in Example !
  • ORF-17 overlapped a partial open reading frame consisting of 542 bp.
  • a search through the nucleic acid and protein databases revealed that the predicted amino acid sequence of this open reading frame was homologous to that of the VirB4, a protein that is associated with virulence in the plant pathogen, A. tumefaciens (Rogowsky et al, "Molecular characterization of the wr regulon oi Agrobacterium tumefaciens: complete nucleotide sequence and gene organization of the 28.63-kbp regulon cloned as a single unit.” Plasmid, 23: 85 (1990); Zupan et al., 1998; Table 1).
  • the open reading frame was therefore designated as ORF-B4.
  • the virB4 gene is part of the virB operon, whose products are involved in the transport of the T-DNA complex from A. tumefaciens to plant cells that subsequently leads to tumor formation.
  • the open reading frame encoding the virB4 gene is 2369 bp long, and is found immediately upstream of the ' r ⁇ 5 gene (Rogowsky et al., 1990).
  • the results of sequencing the pBHIM-2 insert suggest that this is not the case in ⁇ . henselae, in which the gene encoding the 17 kDa antigen is found immediately downstream of the virB4 gene.
  • DNA sequencing of pBHP1 revealed that the upstream region contained two complete open reading frames and one partial open reading frame.
  • the two complete open reading frames were designated as ORF-B2 (SEQ ID NO: 2) and ORF-B3 (SEQ ID NO: 3), which was located downstream from ORF-B2.
  • the sizes of these open reading frames and their encoded products are listed in Table 1.
  • a search through the nucleic acid and protein databases revealed that the predicted amino acid sequences of ORF-B2 (SEQ ID NO: 12) and ORF-B3 (SEQ ID NO: 13) are homologous to those reported for the VirB2 and VirB3 proteins, respectively.
  • the region upstream of the site of hybridization of the probe B4UP in pBHP1 contained the remainder of ORF-B4 that was not present in pBHIM-2, as described in Example 2.
  • EXAMPLE 4 Isolation and Characterization of fi. henselae ORF-15 and virB8 Coding Sequences. Isolation and characterization was performed of nucleic acids at the 3' end of ORF-17 (see Example 1). DNA sequencing and analysis as well as plaque hybridization was performed as described in Example 1.
  • ORF-17 on clone pBHIM-2 is an open reading frame of 966 bp (SEQ ID NO: 6) which was capable of encoding a protein of 321 amino acids (SEQ ID NO: 16) with a molecular weight of 34 kDa.
  • the predicted amino acid sequence of this open reading frame was homologous to that of VirB6, another component of the virB operon of A. tumefaciens (Rogowsky et al., 1995, Table 1). This open reading frame was therefore designated as ORF-B6.
  • ORF-B8 is located 588 bp downstream of ORF-B6.
  • the open reading frame encoding the VirB8 homologue is 713 bp long and lies immediately downstream of the r ⁇ 7 gene.
  • Analysis of the nucleotide sequence of the intergenic region between ORF-B6 and ORF-B8 at both the DNA and protein level did not reveal any homology to the A. tumefaciens virB7 gene.
  • this region did contain an open reading frame (ORF-15) of 411 bp (SEQ ID NO: 7) that was capable of encoding a protein of 136 amino acids (15 kDa) (SEQ ID NO: 17).
  • ORF-15 open reading frame
  • SEQ ID NO: 7 411 bp
  • SEQ ID NO: 17 A BLAST search of the region upstream of ORF-B2 did not reveal the presence of an open reading frame corresponding to a VirB1 homologue. This suggests that the virB1 gene is not present within the cluster of virB genes in B. henselae.
  • primers were designed that were complementary to regions upstream of, and within, ORF-B2. Using these primers, the intervening region from B. henselae genomic DNA was amplified.
  • a digoxigenin-labeled DNA probe complementary to the 3' of the pBHIM-2 insert was synthesized by PCR. This probe, designated as B8DOWN ( Figure 3) (SEQ ID NO: 24), was used in plaque hybridization reactions to isolate recombinant lambda clones from the B. henselae library that contained corresponding sequences.
  • EXAMPLE 5 Isolation and Characterization of fi. henselae virB9, virBIO and virB11 Coding Sequences. Isolation and characterization was performed of nucleic acids in the 3' region of pBHP-2 (see Example 4). DNA sequencing and analysis, as well as plaque hybridization was performed as described in Example 1.
  • EXAMPLE 6 Theoretical Considerations of Genomic Organization Surrounding the 17kDa Antigen Gene of fi. henselae. An analysis was performed of the overall genomic structure surrounding the 17 kDa antigen gene of ⁇ . henselae. DNA sequence analysis was performed as described in Example 1. The assembled sequence derived from the recombinant clones pBHIM-2, pBHP-1 , and pBHP-2 is shown in Figure 1. The assembled sequence was deposited in the GenBank database and assigned the accesssion number AF182718. A major finding of our study is the unexpected genomic localization of the gene encoding the 17 kDa antigen within a cluster of genes homologous to the virB operon of A.
  • ORF-17 The open reading frame encoding the 17 kDa antigen (ORF-17) is located between ORF-B4 and ORF-B6 which encode the VirB4 and VirB6 homologues, respectively.
  • ORF-17 appears to take the place of the open reading frame encoding the VirB ⁇ homologue in A tumefaciens.
  • ⁇ . pertussis another bacterium in which this region has been characterized, the open reading frame encoding the VirB ⁇ homologue is also absent, indicating that ⁇ . henselae is not unique in this respect.
  • ⁇ . pertussis there is no open reading frame between the virB4 and virB6 genes.
  • the presence of the 17 kDa antigen gene between the virB4 and virB6 genes appears to be a feature that is unique to B. henselae.
  • the VirB ⁇ protein is a minor structural component of the virulence pilus that is observed on the surface of vir-induced cells (Zupan, J. R., et al., "Assembly of the VirB transport complex for DNA transfer from Agrobacterium tumefaciens to plant cells," Curr. Opin. Microbiol. 1 : 649 (1998)).
  • the major component of the pilus is the VirB2 protein (Zupan, J.
  • An open reading frame corresponding to the virB7 gene is absent within the virB gene cluster of B. henselae, a property it shares with ⁇ . pertussis. In B. henselae, it is replaced by an open reading frame encoding a 1 ⁇ kDa protein with no significant homology to the VirB7 protein. It is also interesting to note that there is no open reading frame corresponding to the virB1 gene within the virB region of ⁇ . henselae. It is possible that in B. henselde these genes do not play an essential role in the function of the virB locus.
  • genes that are functionally similar to virBI, virB ⁇ , and virB 7 exist in some other part of the chromosome and are expressed independently.
  • Future studies examining the expression of the genes within the virB locus of ⁇ . henselae will help to determine if either of these possibilities is correct. Such studies will also help to elucidate the role of the different virB genes and determine whether this region is involved in the interaction of ⁇ . henselae with host cells, a process that culminates in the pathological lesions of CSD and BA.

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Abstract

L'invention concerne des acides nucléiques de l'opéron de virulence virB de Bartonella henselae et des polypeptides codés par ces acides nucléiques. Elle concerne des vecteurs d'expression contenant lesdits acides nucléiques et des lignées cellulaires transformées contenant lesdits vecteurs d'expression. Elle concerne des méthodes de détection d'une infection induite par B. henselae, et des kits permettant de mettre ces méthodes en oeuvre. Lesdites méthodes consistent à détecter, dans les fluides d'un patient, les acides nucléiques, les polypeptides de l'invention, ou les anticorps agissant contre lesdits polypeptides. Elle concerne des compositions thérapeutiques, telles que des vaccins, permettant de traiter une infection induite par Bartonella henselae. Elle concerne des anticorps orientés contre les polypeptides codés par l'opéron virB de B. henselae. Elle concerne enfin des méthodes permettant de produire des polypeptides codés par l'opéron virB de B. henselae.
PCT/US2000/015465 2000-06-01 2000-06-05 Operon virb de bartonella henselae et proteines codees par cet operon Ceased WO2001092535A1 (fr)

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AU2000253239A AU2000253239A1 (en) 2000-06-01 2000-06-05 Bartonella henselae virb operon and proteins encoded thereby
EP00938158A EP1290177A1 (fr) 2000-06-01 2000-06-05 Operon virb de bartonella henselae et proteines codees par cet operon
CA002414125A CA2414125A1 (fr) 2000-06-01 2000-06-05 Operon virb de bartonella henselae et proteines codees par cet operon

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1454987A1 (fr) * 2003-03-05 2004-09-08 Universität Basel Polypeptides transloqués dans des céllules par le système de sécrétion de type IV VirB/VirD4 et leur utilisations
WO2010021846A3 (fr) * 2008-08-22 2010-05-06 Medical Diagnostic Laboratories, Llc Nouveau polypeptide de 15 kda recombinant et utilisation dudit pour la détection d’une infection par bartonella henselae chez l’humain
EP2326660A4 (fr) * 2008-08-22 2011-09-28 Medical Diagnostic Lab Llc Fragments de recombinaison et peptides synthétiques de polypeptide de 17-kda utilisés dans la détection de bartonella henselae
WO2018183661A1 (fr) * 2017-03-29 2018-10-04 Cornell University Formulation de protection par libération contrôlée de microparticules contenant des vésicules de membrane externe de recombinaison
WO2020010178A1 (fr) * 2018-07-05 2020-01-09 Baylor College Of Medicine Compositions de stimulation immunitaire pour le traitement et la prévention d'infections par des pathogènes intracellulaires

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ANDERSON, B. ET AL.: "Characterization of a 17-Kilodalton Antigen of Bartonella henselae Reactive with Sera from Patients with Cat Scratch Disease", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 33, no. 9, 1995, pages 2358 - 2365, XP000982011 *
FREELAND, R.L. ET AL.: "Identification of Bartonella-Specific Immunodominant Antigens Recognized by the Feline Humoral Immune System", CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, vol. 6, no. 4, July 1999 (1999-07-01), pages 558 - 566, XP000982003 *
SCHMIEDERER, M. ET AL.: "Cloning, Sequencing, and Expression of Three Bartonella henselae Genes Homologous to the Agrobacterium tumefaciens VirB Region", DNA AND CELL BIOLOGY, vol. 19, no. 3, March 2000 (2000-03-01), pages 141 - 147, XP000981946 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1454987A1 (fr) * 2003-03-05 2004-09-08 Universität Basel Polypeptides transloqués dans des céllules par le système de sécrétion de type IV VirB/VirD4 et leur utilisations
WO2010021846A3 (fr) * 2008-08-22 2010-05-06 Medical Diagnostic Laboratories, Llc Nouveau polypeptide de 15 kda recombinant et utilisation dudit pour la détection d’une infection par bartonella henselae chez l’humain
EP2326660A4 (fr) * 2008-08-22 2011-09-28 Medical Diagnostic Lab Llc Fragments de recombinaison et peptides synthétiques de polypeptide de 17-kda utilisés dans la détection de bartonella henselae
EP2328915A4 (fr) * 2008-08-22 2011-09-28 Medical Diagnostic Lab Llc Nouveau polypeptide de 15 kda recombinant et utilisation dudit pour la detection d'une infection par bartonella henselae chez l'humain
CN102203120A (zh) * 2008-08-22 2011-09-28 医疗诊断实验室有限责任公司 新型重组15-kDa多肽及其在人类汉赛巴尔通体感染检测中的用途
US8268569B2 (en) 2008-08-22 2012-09-18 Medical Diagnostic Laboratories, L.L.C. Recombinant 15-kDa polypeptide and use of same in detecting human infection with Bartonella henselae
US8580271B2 (en) * 2008-08-22 2013-11-12 Medical Diagnostic Laboratories, Llc Detection kit containing a novel recombinant 15-kDA polypeptide useful for detecting human infection with Bartonella henselae
US20140141457A1 (en) * 2008-08-22 2014-05-22 Medical Diagnostic Laboratories, Llc DETECTION KIT CONTAINING A NOVEL RECOMBINANT 15-kDA POLYPEPTIDE USEFUL FOR DETECTING HUMAN INFECTION WITH BARTONELLA HENSELAE
WO2018183661A1 (fr) * 2017-03-29 2018-10-04 Cornell University Formulation de protection par libération contrôlée de microparticules contenant des vésicules de membrane externe de recombinaison
WO2020010178A1 (fr) * 2018-07-05 2020-01-09 Baylor College Of Medicine Compositions de stimulation immunitaire pour le traitement et la prévention d'infections par des pathogènes intracellulaires

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