EP1073458A1 - Chlamydia proteine und ihre verwendung - Google Patents

Chlamydia proteine und ihre verwendung

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Publication number
EP1073458A1
EP1073458A1 EP99918748A EP99918748A EP1073458A1 EP 1073458 A1 EP1073458 A1 EP 1073458A1 EP 99918748 A EP99918748 A EP 99918748A EP 99918748 A EP99918748 A EP 99918748A EP 1073458 A1 EP1073458 A1 EP 1073458A1
Authority
EP
European Patent Office
Prior art keywords
seq
chlamydia
infection
amino acid
acid sequence
Prior art date
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Withdrawn
Application number
EP99918748A
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English (en)
French (fr)
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EP1073458A4 (de
Inventor
Daniel D. Rockey
John P. Bannantine
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Oregon State University
Oregon State
Original Assignee
Oregon State University
Oregon State
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Application filed by Oregon State University, Oregon State filed Critical Oregon State University
Publication of EP1073458A1 publication Critical patent/EP1073458A1/de
Publication of EP1073458A4 publication Critical patent/EP1073458A4/de
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C07K14/295—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Chlamydiales (O)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/295—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Chlamydiales (o)

Definitions

  • the present invention relates to the detection of Chlamydia and to the diagnosis, treatment and prevention of Chlamydia infections in animals.
  • Chlamydiae are obligate intracellular bacterial pathogens with a unique biphasic life cycle. They appear as two distinct cellular types, a small dense cell or elementary body (EB) that is enclosed in a rigid bacterial cell wall, and a larger metabolically active reticulate body (RB).
  • the EB is resistant to physical disruption and is infectious, whereas the RB is more fragile and only exists inside cells.
  • the Chlamydia life cycle begins with the attachment of the EB form to the host cell which is followed by endocytosis into a nascent vacuole, also called an "inclusion membrane. " After EB attachment and entry, replication of the EB form produces RB forms that continue to grow within the vacuole.
  • Chlamydia includes two species that are primarily associated with human disease: C. trachomatis and C. pneumoniae.
  • C. trachomatis causes trachoma, an eye disease that is the leading cause of preventable infectious blindness worldwide with an estimated 500 million cases of active trachoma worldwide.
  • C. trachomatis also causes a sexually transmitted chlamydial disease which is very common worldwide.
  • C. trachomatis also causes lymphogranuloma venereum, a debilitating systemic disease characterized by lymphatic gland swelling.
  • the most serious sequelae of chlamydial genital infections of females include salpingitis, pelvic inflammatory disease, and ectopic pregnancy.
  • the World Health Organization estimates that 89 million new cases of genital Chlamydia occurred worldwide in 1995 (Peeling and Brunham, 1996).
  • pneumoniae causes respiratory diseases including so called walking pneumonia, a low- grade disease such that the infected person frequently fails to obtain treatment and remains in the community as an active, infectious carrier.
  • C. pneumoniae is currently of interest because of its strong epidemiological association with coronary artery disease, and there is also some evidence to link it with multiple sclerosis.
  • Chlamydia psittaci and Chlamydia pecorum are primarily pathogens of wild and domestic animals, but these species may infect -2-
  • C. psittaci is acquired through respiratory droplet infection and is considered an occupational health hazard for bird fanciers and poultry workers.
  • Chlamydia polypeptides for use as vaccines that induce a protective immune response without inducing the pathological response caused by the antigens associated with the EB form of Chlamydia.
  • Such immunostimulatory peptides will be useful in the treatment, as well as in the diagnosis, detection and prevention of Chlamydial infections.
  • the present invention includes the use of Chlamydia proteins that show enhanced expression in the reticulate body (RB) stage relative to the elementary body (EB) stage of the Chlamydia life cycle. These proteins are not present at detectable levels in the EB form using current immunological techniques and are thus said to be “infection-specific. " Certain of these infection-specific proteins are found in the inclusion membrane of the infected cell, and so have been termed "Inc” proteins. These include the IncA, IncB, and IncC proteins of Chlamydia as described in the present disclosure. The genes mat encode the IncA, IncB and IncC proteins are referred to as inc A, incB and incC respectively.
  • TroA and TroB proteins have been so named because they resemble the Tro proteins of Treponema pallidum, which are thought to form part of an ABC transport system.
  • the infection-specific Chlamydia proteins of the disclosure are recognized by convalescent antisera (i.e. , antisera taken from an animal that has recovered from a Chlamydia infection) but are not recognized by antisera against the killed EB form of Chlamydia.
  • convalescent antisera i.e. , antisera taken from an animal that has recovered from a Chlamydia infection
  • antisera against the killed EB form of Chlamydia are expressed only during active chlamydial infection and are therefore useful as protective antigens.
  • These infection-specific proteins may be used to confer a protective immune response without inducing a pathological effect.
  • immuno- fluorescence microscopy and immunoblotting with antisera demonstrated that the infection-specific proteins are present in Chlamydia-infecte ⁇ HeLa cells, but are undetectable in purified EBs and absent in uninfected HeLa cells.
  • Immunofluorescense microscopy reveals that IncA, IncB and IncC are localized to the inclusion membrane of infected HeLa cells.
  • Reverse-transcription polymerase chain reactions (RT-PCR), northern hybridization data, and restriction analysis revealed that the incB and incC genes are closely linked and transcribed in an operon.
  • the present invention includes the nucleotide and amino acid sequences for certain infection-specific proteins from Chlamydia. These proteins are p242, TroA, and TroB from C. trachomatis, and the IncB, and IncC proteins from C. psittaci.
  • the scope of the invention includes fragments of these proteins that may be used in a vaccine preparation or that may be used in a method of detecting Chlamydia antibodies. Such fragments may be, for example, 5, 10, 15, 20, 25, or 30 contiguous amino acids in length. They may even encompass the entire protein.
  • the present invention encompasses the purified infection-specific proteins having amino acid sequences as shown in SEQ ID NOS: 2, 4, 6, 10, and 12, amino acid sequences mat differ from such sequences by one or more conservative amino acid substitutions, and amino acid sequences that show at least 75 % sequence identity with such amino acid sequences.
  • invention also includes isolated nucleic acid molecules that encode a protein as described in the above paragraph, including isolated nucleic acid molecules with nucleotide sequences as shown in SEQ ID NOS: 1 ,3, 5, 9, and 11.
  • the present invention also includes a vaccine or immunostimulatory preparation directed against the reticulate body (RB) form of Chlamydia comprising one or more purified infection- specific peptides (or portions or fragments thereof, or peptides showing sequence similarity to a portion of such a peptide).
  • RB reticulate body
  • Such peptide fragments may be, for example, 5, 10, 15, 20, 25, or 30 contiguous amino acids in length, of the sequence shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, or 18.
  • Peptides used in such a vaccine may even encompass the entire purified peptide of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, or 18, a peptide that differs from such a peptide by one or more conservative amino acid substitutions, or a peptide having at least 75 % sequence identity with such a peptide.
  • Such vaccine preparations may contain one or more pharmaceutically acceptable excipients, adjuvants or diluents.
  • the invention additionally encompasses methods for making a vaccine, comprising combining a pharmaceutically acceptable excipient with a peptide described herein. Also included is a method of vaccination comprising administering a vaccine as described herein to a mammal.
  • the present invention also provides a method for the diagnostic use of the disclosed purified infection-specific peptides, for instance by use in a diagnostic assay to detect the presence of infection-specific antibodies in a medical specimen, in which antibodies bind to the Chlamydia peptide and indicate that the subject from which the specimen was removed was previously -4-
  • Such a method may comprise: (i) supplying a biological sample, such as blood from an animal, that is suspected to contain infection-specific zn ⁇ -Chlamydia antibody, (ii) contacting the sample with at least one infection-specific Chlamydia peptide described herein, such that a reaction between the peptide and the infection-specific mti-Chlamydia antibody gives rise to a detectable effect, such as a chromogenic conversion; and (iii) detecting this detectable effect.
  • the present invention also provides a method of using antibodies that bind specifically with the disclosed proteins for detection of infection-specific Chlamydia antigen, indicating the presence of Chlamydia in the RB stage as distinct from the EB stage.
  • the relevant infection-specific antibodies may be used to provide specific binding in an Enzyme Linked Immunosorbant Assay (ELISA) or other immunological assay wherein the antibody Fc portion is linked to a chromogenic, fluorescent or radioactive molecule and the Fab portion specifically interacts with, and binds to, an infection-specific protein.
  • ELISA Enzyme Linked Immunosorbant Assay
  • Such a method may comprise: (i) supplying a biological sample from an animal suspected to contain an infection-specific Chlamydia antigen, and (ii) contacting the sample with at least one infection-specific anti- Chlamydia antibody, such that a reaction between the antibody and the infection-specific Chlamydia protein gives rise to a detectable effect; and (iii) detecting this detectable effect.
  • probes and primers derived from the nucleotide sequences that encode infection-specific peptides, to detect the presence of Chlamydia nucleic acids in medical specimens.
  • probes and primers may be nucleotide fragments, of, for example, 15, 20, 25, 30 or 40 contiguous nucleotides of the sequence shown in SEQ ID NOS: 1 , 3, 5, 7, 9, 11 , 13, 15, or 17.
  • An additional aspect of the invention is a method of treating a Chlamydia infection by directing a therapeutic agent against a specific target, where the target is chosen from an infection specific protein of Chlamydia, a gene that encodes an infection-specific protein of Chlamydia, and an RNA transcript that encodes an infection-specific protein of Chlamydia, wherein the therapeutic agent interacts with said target to affect a reduction in pathology.
  • SEQ ID NO: l shows a nucleic acid sequence encoding the p242 C. trachomatis protein, with deduced primary amino acid sequence also shown.
  • SEQ ID NO: 2 shows the amino acid sequence of the p242 C. trachomatis protein.
  • SEQ ID NO: 3 shows a nucleic acid sequence encoding the TroA C. trachomatis protein, with deduced primary amino acid sequence also shown.
  • SEQ ID NO:4 shows the amino acid sequence of the TroA C. trachomatis protein.
  • SEQ ID NO: 5 shows a nucleic acid sequence encoding the TroB C. trachomatis protein, with deduced primary amino acid sequence also shown. -5-
  • SEQ ID NO:6 shows the amino acid sequence of the TroB C. trachomatis protein.
  • SEQ ID NO: 7 shows a nucleic acid sequence encoding the IncA C. psittaci protein, with deduced primary amino acid sequence also shown.
  • SEQ ID NO:8 shows the amino acid sequence of the IncA C. psittaci protein.
  • SEQ ID NO: 9 shows a nucleic acid sequence encoding the IncB C. psittaci protein, with deduced primary amino acid sequence also shown.
  • SEQ ID NO: 10 shows the amino acid sequence of the IncB C. psittaci protein.
  • SEQ ID NO: 11 shows a nucleic acid sequence encoding the IncC C. psittaci protein, with deduced primary amino acid sequence also shown.
  • SEQ ID NO: 12 shows the amino acid sequence of the IncC C. psittaci protein.
  • SEQ ID NO: 13 shows a nucleic acid sequence encoding the IncA C. trachomatis protein, with deduced primary amino acid sequence also shown.
  • SEQ ID NO: 14 shows the amino acid sequence of the IncA C. trachomatis protein.
  • SEQ ID NO: 15 shows a nucleic acid sequence encoding the IncB C. trachomatis protein, with deduced primary amino acid sequence also shown.
  • SEQ ID NO: 16 shows the amino acid sequence of the IncB C. trachomatis protein.
  • SEQ ID NO: 17 shows a nucleic acid sequence encoding the IncC C. trachomatis protein, with deduced primary amino acid sequence also shown.
  • SEQ ID NO: 18 shows the amino acid sequence of the IncC C. trachomatis protein.
  • SEQ ID NO: 19 shows the upstream oligonucleotide used to amplify the C. psittaci incC
  • SEQ ID NO:20 shows the downstream oligonucleotide used to amplify the C. psittaci incC ORF.
  • SEQ ID NO:21 shows the upstream oligonucleotide used to amplify the C. psittaci incB ORF.
  • SEQ ID NO:22 shows the downstream oligonucleotide used to amplify the C. psittaci incB ORF.
  • SEQ ID NO:23 shows the upstream oligonucleotide used to amplify the C. psittaci inch ORF.
  • SEQ ID NO: 24 shows the downstream oligonucleotide used to amplify the C. psittaci inch ORF.
  • EB refers to the Elementary Body, an environmentally refractile and largely metabolically dormant form of Chlamydia that is infectious and is presented as a small dense body enclosed by a bacterial cell wall.
  • RB refers to the Reticulate Body, a metabolically active form of Chlamydia that is not infectious, and exists only within a host cell, being very fragile, often branched, and appearing larger and less dense that the EB.
  • Infection-specific refers to a protein that shows enhanced expression in the RB form of Chlamydia compared to the EB form. Infection-specific proteins are not necessarily absent from the EB form, but they are significantly more common in the RB form than in the EB form. "infection-specific antibody” is an antibody that binds specifically to an infection-specific protein.
  • Bio sample refers to any sample of biological origin including, but not limited to a blood sample, a plasma sample, a mucosal smear or a tissue sample.
  • isolated nucleic acid has been substantially separated or purified away from other nucleic acid sequences in the cell of the organism in which the nucleic acid naturally occurs, i.e. , other chromosomal and extrachromosomal DNA and RNA.
  • isolated thus encompasses nucleic acids purified by standard nucleic acid purification methods. The term also embraces nucleic acids prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • Probes and “primers. " Nucleic acid probes and primers may readily be prepared based on the nucleic acid sequences provided by this invention.
  • a "probe” comprises an isolated nucleic acid attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
  • Primer pairs can be used for amplification of a nucleic acid sequence, e.g. , by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art.
  • Probes and primers as used in the present invention typically comprise at least 15 nucleotides of the nucleic acid sequences that are shown to encode infection-specific proteins.
  • probes and primers may also be employed, such as probes and primers that comprise at least 20, 30 or 40 consecutive nucleotides of the disclosed nucleic acid sequences.
  • Methods for preparing and using probes and primers are well known in the art and are described in, for example Sambrook et al. (1989); Ausubel et al. , (1987); and Innis et al. , (1990).
  • PCR primer pairs can be derived from a known sequence, for example, by using computer -7-
  • Constant amino acid substitutions are those substitutions that, least interfere with the properties of the original protein, i.e. , the structure and especially the function of the protein is conserved and not significantly changed by such substitutions.
  • the table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative substitutions.
  • Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • substitutions which in general are expected to produce the greatest changes in protein properties will be non-conservative, for instance changes in which (a) a hydrophilic residue, e.g. , seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. , leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, e.g.
  • a hydrophilic residue e.g. , seryl or threonyl
  • a hydrophobic residue e.g. , leucyl, isoleucyl, phenylalanyl, valyl or alany
  • sequence identity The similarity between two nucleic acid sequences, or two amino acid sequences is expressed in terms of the level of sequence identity shared between the sequences. Sequence identity is typically expressed in terms of percentage identity; the higher the percentage, the more similar the two sequences are.
  • Variants of naturally occurring infection- specific peptides useful in the present invention are typically characterized by possession of at least 50% sequence identity counted over the full length alignment with the amino acid sequence of a naturally occurring infection-specific peptide when aligned using BLAST 2.0.1 (Altschul et al. , 1997).
  • NCBI National Center for Biotechnology Information
  • blastp blastn
  • blastx blastx
  • tblastn tblastx
  • It can be accessed at http//www. ncbi.nlm.nih.gov/BLAST/.
  • a description of how to determine sequence identity using this program is available at http : //www . ncbi . nlm . nih . gov/BLAST/blast help . html .
  • Variants of namrally occurring infection-specific nucleic acid sequences useful in the present invention are typically characterized by possession of at least 50% sequence identity counted over the full length alignment with the nucleic acid sequence of a naturally occurring infection-specific ORF when aligned using BLAST 2.0.1.
  • Useful nucleic acids may show even greater percentage identity, and may, for example, possess at least 55% , at least 65 % , at least 75 %, at least 80% , at least 85 %, at least 90% , or at least 95 % sequence identity naturally occurring infection-specific ORF.
  • a first nucleic acid sequence is “operably” linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • Recombinant A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
  • Stringent Conditions Stringent conditions, in the context of nucleic acid hybridization, are sequence-dependent and are different under different environmental parameters. Generally, stringent conditions are selected to be about 5 degrees to 20 degrees lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • the Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Conditions for nucleic acid hybridization and calculation of stringencies can be found in Sambrook et al. (1989), pages 9.49-9.55.
  • Typical high stringency hybridization conditions may include, for example, wash conditions of 0.1 X SSC, 0.5% SDS at a wash temperature of 68 ° C .
  • the term "specific for (a target sequence)" indicates that the probe or primer hybridizes under high-stringency conditions substantially only to the target sequence in a given sample comprising the target sequence.
  • a purified peptide is a peptide that has been extracted from the cellular environment and separated from substantially all other cellular peptides.
  • the term peptide includes peptides, polypeptides and proteins.
  • a purified peptide is a preparation in which the subject peptide comprises 50% or more of the protein content of the preparation. For certain uses, such as vaccine preparations, even greater purity may be preferable.
  • Immunomulatory peptide refers to a peptide that is capable of stimulating a humoral or antibody-mediated immune response when inoculated into an animal.
  • Vaccine A vaccine is a composition containing at least one immunostimulatory peptide which may be inoculated into an animal with the intention of producing a protective immunological reaction against a certain antigen.
  • the antigen to be protected against may be, for instance, an infectio-specific antigen of Chlamydia.
  • Chlamydia (C. psittaci strain GPIC or C. trachomatis LGV-434, ser. L2) was cultivated in HeLa 229 cells using standard methods (Caldwell et al., 1981). Purified Chlamydiae were obtained using Renografin (E. R. Squibb & Sons, Inc., Princeton, N.J.) density gradient centrifugation. Escherichia coli DH5D (Bethesda Research Laboratories, Inc.,
  • E. coli XLl-Blue MRF' (Stratagene, La Jolla, Calif.) was used as the host strain for infection with lambda ZAPII phage vector.
  • E. coli SOLR (Stratagene) was used as the host strain for infection with in vivo excised filamentous lambda ZAPII. -10-
  • MBP Maltose Binding Protein
  • Inc fusion proteins were used as antigens for the production of mono-specific antibody reagents in Hartley strain guinea-pigs.
  • the protein was diluted to 100 ⁇ g/ml "1 sterile saline and mixed wifii the Ribi Trivalent Adjuvant (Ribi Immunochem.).
  • the antigen/adjuvant emulsion was administered to anaesthetized guinea-pigs using a procedure provided by the manufacturer. Sera were collected 14 days after secondary and tertiary immunizations.
  • Control antisera were produced by immunizing guinea-pigs with adjuvant alone, or with adjuvant plus purified maltose-binding protein.
  • Convalescent guinea-pig antisera, antisera against live EBs, and antisera against formalin- fixed EBs were produced using standard methods (Rockey and Rosquist, 1994 and Rockey et al. , 1995).
  • C. psittaci library construction and screening For the incB and incC genes, C. psittaci strain GPIC DNA was extracted using a genomic DNA extraction kit (Qiagen) with one modification; dithiofhreitol (5mM) was added to the suspension buffer to assist EB lysis. DNA was partially digested with Tsp509I and ligated to EcoRl digested ⁇ -ZAPII phage arms (Stratagene). The ligation was packaged in vitro with Gigapack extracts according to the manufacturer's instructions (Stratagene). Recombinant phage were plated on E. coli XL-1 Blue at densities of approximately 10 4 PFU/150-mm (diameter) plate.
  • genomic DNA from C. psittaci strain GPIC was partially digested with Sau3A, size-selected (2-8 kb) by electrophoresis through low-melting-temperature agarose, and blunt-ended with T4 DNA polymerase.
  • This DNA was ligated to an EcoRl/Notl adapter (Life Technologies), kinased, and ligated to EcoRl -digested Lambda ZAP II vector (Stratagene Cloning Systems). Recombinants were packaged (Lambda Gigapack Gold, Stratagene) and used to infect E. coli XLl-Blue (Stratagene). Plaques were allowed to develop for 4 h at 37°C.
  • Nitrocellulose filters laden with 10 mM IPTG were placed onto the plaques and incubated for an additional 4 h at 37 °C. These filters were removed and placed into a blocking solution consisting of PBS (150 mM NaCl, 10 mM NaPO, pH7.2) plus 0.1 % Tween-20 (TPBS) and 2% BSA-TPBS. Filters were incubated for 1 h, rinsed twice in TPBS, and incubated overnight in convalescent-guinea-pig sera diluted 1: 100 in BSA-TPBS. After three washes in TPBS, the filters were incubated for 1 h in I25 l-staphylococcal protein A (New England Nuclear) diluted to approx.
  • PBS 150 mM NaCl, 10 mM NaPO, pH7.2
  • TPBS 0.1 % Tween-20
  • the plasmid pJC2 contains a 5.0 kb EcoRl GPIC genomic fragment cloned into the pZEro2.1 vector (Invitrogen).
  • the incC ORF sequence was 32 P-radiolabeled using random priming (Gibco-BRL) and used to probe £coRI cut GPIC genomic DNA fragments separated by agarose gel electrophoresis. Fragments in the size range of the positive signal were excised from the gel and purified by Gene- Clean (BiolOl). The gel-purified fragments were used in a ligation along with EcoRI-digested pZEro2.1. Kanamycin resistant colonies were screened by colony hybridization with radiolabeled incC.
  • MBP fusions of the five ORFs present in pJC2 were produced using the pMAL-C2 vector (New England Biolabs).
  • the upstream and downstream oligonucleotides for this amplification were
  • the vector was digested with Xmnl and BamRl, and the amplication product was digested with Bam ⁇ l. Ligation of these products resulted in an in-frame fusion between the malE gene in the vector and the incC reading frame from pBS200-7. The stop codon for this construction is provided by the insert. Following ligation, the products were transformed into E.coli strain HD5D. The resulting fusion protein (MBP/IncC) was overexpressed and purified by maltose affinity chromatography using an amylose resin supplied by New England Biolabs.
  • MBP/IncB and MBP/IncC fusion proteins were used as antigen for the production of monospecific antibody in Hartley strain guinea-pigs by standard methods (Rockey et al., 1995). Inserts in each construct were confirmed by DNA sequencing. -12-
  • a maltose-binding protein/IncA fusion protein was produced using the pMAL- C2 vector system from New England Biolabs.
  • the upstream and downstream oligo- nucleotides for this amplification were
  • the vector was digested with Xmn and Bam ⁇ , and the amplification product was digested with Seal and BamH . Ligation of these products resulted in an in-frame fusion between the malE gene in the vector and the inc A reading frame from pGP17. The stop codon for this construction is provided by the insert. Following ligation, the products were transformed into E. coli strain DH5D. The resulting fusion protein (MBP/IncA) was overexpressed and purified by maltose affinity chromatography using amylose resin (New England Biolabs).
  • MBP/IncA was used as antigen for the production of mono-specific antibody reagents in Hartley strain guinea-pigs.
  • nucleotide sequencing was conducted using the Sequences system (US Biochemical) with the M13 forward and reverse primers, and internal primers synthesized on an Milligen/Biosearch Cyclone Plus DNA synthesizer.
  • Computer analyses were conducted using the MacVector Sequence Analysis Software (International Biotechnologies Incorporated).
  • Hydrophilicity profiles were determined using the Kyte-Doolittle scale (Kyte and Doolittle, 1982) with a window of 7. Secondary-structure predictions were generated using a combination of the
  • RNA for RT-PCR analysis was extracted from approximately 2 x 10 14 C. psittaci-mfected cells. A Qiagen column was used for extraction and purification according to the manufacturer's instructions (Qiagen). RQ1 RNase DNase (Promega) was used to ensure removal of contaminating genomic DNA. cDNA was prepared by incubating 1.5 ⁇ g of RNA, 2.5 ⁇ M of the reverse oligonucleotide primer, and AMV reverse transcriptase (Promega) for 1 hour at 42°C in sodium pyrophosphate buffer, according to the manufacturer's instructions.
  • PCR reactions were carried out using 1 ⁇ l of the cDNA reaction, 1.25 ⁇ M of each oligonucleotide primer, and Pwo polymerase (Boehringer Mannheim). Each RT-PCR reaction was accompanied by a positive control reaction that utilized the same primer set and 10 ng of C. psittaci genomic DNA, and a negative control reaction in which 1 ⁇ l of the same RNA preparation was used as template in the PCR reaction. A control primer set located within the incC gene was also used as an RT-PCR control.
  • E. coli SOLR (Stratagene) was used as the host strain for infection with in vivo excised filamentous lambda ZAPII.
  • Sera were collected every two weeks and anti-chlamydial titers were determined. These animals showed mild clinical signs of disease which cleared spontaneously. A second challenge was then administered. Sera were collected from these animals and used to probe a C. trachomatis expression library as discussed below. As a control, Guinea Pigs were immunized with killed C. trachomatis of the EB form. Sera from these animals were obtained and also used to probe the C. trachomatis expression library.
  • a C. trachomatis genomic library was constructed with the lambda ZAPII vector as described above for C. psittaci. Approximately 15,000 plaques were plated, transferred to nitrocellulose filters (Schleicher and
  • coli showed that each expressed one or more proteins that reacted with convalescent (anti-RB) antisera but not with the anti-EB antiserum.
  • pCt2 and pCt3 expressed an identical 19.9 kDa protein (p242).
  • the pCt4 recombinant expressed two different proteins of approximately 32 kDa each that are strongly recognized by convalescent antisera (TroA and TroB).
  • the insert in pCt4 contains two complete ORFs which code for two proteins, each of approximately 32kDa (TroA and TroB) that show some homology with proteins from Treponema -15-
  • the present invention includes the nucleotide and amino acid sequences for certain infection-specific proteins from Chlamydia. These proteins are p242, TroA, and TroB from C. trachomatis, and the IncB, and IncC proteins from C. psittaci.
  • the scope of the invention includes fragments of fhese proteins that may be used in a vaccine preparation or that may be used in a method of detecting Chlamydia antibodies. Such fragments may be, for example, 5, 10, 15, 20, 25, or 30 contiguous amino acids in length, or may even encompass the entire protein.
  • the present invention also encompasses the use of infection-specific proteins of Chlamydia, and the use of nucleotides encoding such proteins.
  • Infection-specific proteins include the IncA, IncB and IncC proteins of C. psittaci, the IncA, IncB and IncC proteins of C. trachomatis, and the TroA, TroB, and p242 proteins of C. trachomatis.
  • the inventors have shown that these proteins are infection-specific by using immunological techniques such as immuno- fluorescence microscopy and immunoblotting.
  • the present invention includes a vaccine against chlamydial infections comprising infection-specific proteins or fragments of these proteins or proteins that are homologous or show substantial sequence similarity to fhese proteins.
  • one or more purified infection-specific proteins may be mixed with a pharmaceutically acceptable excipient to produce a vaccine that stimulates a protective immunological response in an animal.
  • the vaccine may be administered intra-muscularly or sub-cutaneously or intravenously.
  • the vaccine may be administered by inoculation into or onto the mucous membranes of the subject animal.
  • the vaccine may be administered urethrally or genitally as a liquid or in the form of a pessary. In another embodiment, it may be administered to the mucosa of the lungs as a spray or vapor suspension.
  • the vaccine should comprise at least three consecutive amino acids, preferably at least five consecutive amino acids, and may comprise at least 10, 15, 25, 30, 40, or 45 consecutive amino acids of the infection-specific proteins as shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, and 18.
  • the vaccine of the invention may be used to inoculate potential animal targets of any of the chlamydial diseases including those caused by C. psittaci, C. trachomatis, C. pneumoniae or C. pecorum. Indeed the vaccine of the invention may be used to inoculate animals against any disease that shows immunological cross-protection as a result of exposure to infection-specific Chlamydia antigen.
  • Vaccines of the present invention can include effective amounts of immunological adjuvants known to enhance an immune response (e.g. , alum).
  • the protein or polypeptide is present in the vaccine in an amount sufficient to induce a protective immune response whether -16-
  • the present invention also teaches a method of making a vaccine against chlamydial infections.
  • the method of making the vaccine comprises providing a pure (or substantially pure) infection-specific chlamydial peptide or portion thereof, and mixing the peptide with a pharmacologically acceptable excipient or adjuvant.
  • Adjuvants may include commonly used compounds such as alum.
  • the vaccines may be formulated using a peptide according to the present invention together with a pharmaceutically acceptable excipient such as water, saline, dextrose and glycerol.
  • the vaccines may also include auxiliary substances such as emulsifying agents and pH buffers. Doses of the vaccine administered will vary depending on the antigenicity of the particular peptide or peptide combination employed in the vaccine and characteristics of the animal or human patient to be vaccinated.
  • the infection-specific vaccine of the invention is directed towards not only C. psittaci, but against all forms of Chlamydia including C. pneumoniae, C. trachomatis and C. pecorum, and the vaccine may comprise not just peptides derived from C.
  • the present invention also teaches a method of vaccination comprising administering a vaccine formulated as described above to an animal either intravenously, intramuscularly, subcutaneously, by inhalation of a vapor or mist, or by inoculation in the form of a liquid, spray, ointment, pessary or pill into or onto the mucous membranes of the mouth, nose, lungs or urogenital tract or colon.
  • a vaccine formulated as described above to an animal either intravenously, intramuscularly, subcutaneously, by inhalation of a vapor or mist, or by inoculation in the form of a liquid, spray, ointment, pessary or pill into or onto the mucous membranes of the mouth, nose, lungs or urogenital tract or colon.
  • the methods of the invention may be practiced equally with human or non-human animal subjects.
  • the present invention also teaches a method of detecting Chlamydia infection-specific proteins produced by the Reticulate Body form of the organism.
  • antibodies raised to the infection-specific proteins are used in an immunological assay such as an Enzyme Linked Immunosorbant Assay or Biotin-Avidin assay or a radioimmunoassay or any other assay wherein specific antibodies are used to recognize a specific protein.
  • an immunological assay such as an Enzyme Linked Immunosorbant Assay or Biotin-Avidin assay or a radioimmunoassay or any other assay wherein specific antibodies are used to recognize a specific protein.
  • Such assays may be used to detect both the quantity of proteins present and also the specificity of binding of such proteins.
  • antibodies have attached to them, usually at the Fc portion, a detectable label, such as an enzyme, fluorescent marker, a radioactive marker or a Biotin-Avidin system marker that allows detection.
  • a biological sample is provided from an animal that has been putatively exposed to Chlamydia.
  • a sample may be, for example, whole blood, serum, tissue, saliva or a mucosal secretion.
  • the sample is then contacted with the labeled antibody and specific binding, if any, is detected.
  • antigens that are present in cells or tissues include immunofluorescense, indirect- immunofluorescense and immunohistochemistry.
  • immunofluorescense a fluorescent dye is bound directly to the antibody.
  • indirect-immunofluorescence the dye is bound to an anti- immunoglobulin. Specific binding occurs between antigen and bound antibody is detected by virme of flourescent emissions from the dye moiety. This technique would be particularly useful, for instance, for detection of Chlamydia antigen present on a urogenital mucosal smear.
  • the present invention also teaches a method of detection of Chlamydia infection-specific antibodies made against the Reticulate Body.
  • a sample is provided from an animal putatively exposed to Chlamydia to determine whether the sample contains infection- specific antibodies.
  • a sample may be, for example, whole blood, serum, tissue, saliva or a mucosal secretion.
  • This sample is contacted with infection-specific antigens such that the amount and specificity of binding of the antibody may be measured by its binding to a specific antigen.
  • Many techniques are commonly known in the art for the detection and quantification of antigen.
  • the purified antigen will be bound to a substrate, the antibody of the sample will bind via its Fab portion to this antigen, the substrate will then be washed and a second, labeled antibody will then be added which will bind to the Fc portion of the antibody that is the subject of the assay.
  • the second, labeled antibody will be species specific, i.e. , if the serum is from a human, the second, labeled antibody will be anti-human- IgG antibody.
  • the specimen will then be washed and the amount of the second, labeled antibody that has been bound will be detected and quantified by standard methods.
  • the present invention also teaches a method of treating a Chlamydial infection by directing a therapeutic agent against a specific target, such as: (i) an infection-specific protein of Chlamydia, (ii) a gene that encodes an infection-specific protein of Chlamydia and (iii) an RNA transcript that encodes an infection-specific protein of Chlamydia, wherein said therapeutic agent interacts with said target to affect a reduction in pathology.
  • a specific target such as: (i) an infection-specific protein of Chlamydia, (ii) a gene that encodes an infection-specific protein of Chlamydia and (iii) an RNA transcript that encodes an infection-specific protein of Chlamydia, wherein said therapeutic agent interacts with said target to affect a reduction in pathology.
  • a specific target such as: (i) an infection-specific protein of Chlamydia, (ii) a gene that encodes an infection-specific protein of Chlamyd
  • RNA molecules complementary to transcripts of infection specific genes are introduced into the host cells that contain Chlamydia, and by binding to the mRNA transcripts of the infection-specific genes, prevent translation and therefore expression of the infection-specific proteins that are associated with pafhogenesis.
  • the invention may be practiced to produce a vaccine against any species of Chlamydia, including C. psittaci, C pecorum, C. trachomatis and C. pneumoniae. -18-
  • SEQ ID NOS: 1-18 The DNA and protein sequences discussed herein are shown in SEQ ID NOS: 1-18. These sequences refer to infection-specific proteins and to the DNA sequences that encode fhese proteins. Although these sequences are from C. psittaci and C. trachomatis, it would be equally possible to substitute in the present invention, the orfhologs of these sequences from other Chlamydia species such as C. pecorum and C. pneumoniae.
  • orthologous sequences may be obtained from the appropriate organisms by isolation of the genome of the organism, digestion with restriction enzymes, separation of restriction fragments by electrophoresis and purification of these fragments and selection of fragments of appropriate size. Identity of the fragments can be confirmed by dot-blot and by standard DNA sequencing techniques.
  • the orthologous sequences in different Chlamydia species may also be found by selection of appropriate PCR primers (selected from appropriate regions flanking the Chlamydia gene of interest), and the use of these primers in a PCR reaction, using the genome of the particular species of Chlamydia of interest as a template, to amplify the orfholog of interest.
  • Such PCR primers would be selected from the flanking regions to allow specific amplification of the target gene.
  • the fragments so obtained could then be run on a gel to check size and sequenced and compared against the known sequences to determine sequence identity.
  • the degree of sequence identity between the infection-specific genes of C. psittaci or C. trachomatis and their orfhologs from C. pecorum and C. pneumoniae may be determined by comparing sequences using the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) as described herein.
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local Alignment Search Tool
  • Orthologues of interest infection-specific proteins are characterized by possession of at least 50% or greater sequence identity counted over the full length alignment with one of the disclosed amino acid sequences of the C. psittaci or C. trachomatis infection-specific proteins using gapped blastp set to default parameters (described herein).
  • EXAMPLE 2 Heterologous Expression of Infection-Specific Antigens Methods for expressing large amounts of protein from a cloned gene introduced into
  • Escherichia coli may be utilized for the purification of the Chlamydia peptides.
  • Methods and plasmid vectors for producing fusion proteins and intact native proteins in bacteria are well known and are described in Sambrook et al. (1989). Such fusion proteins may be made in large amounts, are relatively simple to purify, and can be used to produce antibodies.
  • Native proteins can be produced in bacteria by placing a strong, regulated promoter and an efficient ribosome binding site upstream of the cloned gene. If low levels of protein are produced, additional steps -19-
  • Vectors suitable for the production of intact native proteins include pKC30 (Shimatake and Rosenberg (1981)), pKK177-3 (Amann and Brosius (1985)) and pET-3 (Studiar and Moffatt
  • Fusion proteins may be isolated from protein gels, lyophilized, ground into a powder and used as antigen preparations.
  • Mammalian or other eukaryotic host cells such as those of yeast, filamentous fungi, plant, insect, amphibian or avian species, may also be used for protein expression, as is well known in the art.
  • Examples of commonly used mammalian host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cells, and WI38, BHK, and COS cell lines, although it will be appreciated by the skilled practitioner that other prokaryotic and eukaryotic cells and cell lines may be appropriate for a variety of purposes, e.g. , to provide higher expression, post-translational modification, desirable glycosylation patterns, or other features.
  • peptides particularly shorter peptides, may be chemically synthesized, avoiding the need for purification from cells or culture media. It is known that peptides as short as
  • peptides can act as an antigenic determinant and stimulate an immune response.
  • Such peptides may be administered as vaccines in ISCOMs (Immune Stimulatory Complexes) as described by Janeway & Travers, Immunobiology: The Immune System In Health and Disease,
  • one aspect of the present invention includes small peptides encoded by the nucleic acid molecules disclosed herein.
  • Such peptides include at least 5, and may be at least 10, 15, 20, 25, or 30 or more contiguous amino acids of the polypeptide sequences described herein.
  • Antibody against infection-specific antigen is encompassed by the present invention, particularly for the detection of Chlamydia infection-specific antigen.
  • Such antibody may be produced by inoculation of an animal such as a guinea-pig or a monkey with infection-specific antigen produced as described above.
  • antigen may be a polypeptide as disclosed herein, such as a complete or partial polypeptide from C. psittaci, C. trachomatis, C. pneumoniae or C. pecorum. As discussed above, any molecule that can elicit a specific, protective immune response -20-
  • a vaccine may be used as a vaccine, but since a minimum of three amino acids are required to do this, a vaccine should comprise at least three amino acids.
  • the peptide for use in the vaccine of the invention may be naturally derived or may be synthetic such as those synthesized on a commercially available peptide synthesizer.
  • the peptide may also comprise a complete or partial peptide derived from the C. pneumoniae or C. pecorum infection-specific orfhologs of the C. trachomatis or C. psittaci proteins as set out herein.
  • a polyclonal antibody is produced by providing a purified peptide which is diluted to 100 micrograms per milliliter in sterile saline and mixed with RiBi Trivalent Adjuvant (RiBi Immunochem Inc). The antigen/adjuvant emulsion is then administered to an anaesthetized guinea pig using a procedure as provided by the manufacturer. Serum is collected 14 days after secondary and tertiary immunizations.
  • Monoclonal antibody to epitopes of the Chlamydia peptides identified and isolated as described can be prepared from murine hybridomas according to the classical method of Kohler and Milstein (1975) or derivative methods thereof. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected purified protein over a period of a few weeks. The mouse is then sacrificed, and the antibody-producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin, e.g. , Hypoxanthene, Aminopterin and Thymidine (HAT) medium. The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued.
  • aminopterin e.g. , Hypoxanthene, Aminopterin and Thy
  • Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall (1980), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Harlow and Lane (1988).
  • An alternative approach to raising antibodies against the Chlamydia peptides is to use synthetic peptides synthesized on a commercially available peptide synthesizer based upon the amino acid sequence of the peptides predicted from nucleotide sequence data.
  • monoclonal antibodies that recognize a specific Chlamydia peptide are produced.
  • monoclonal antibodies will be specific to each peptide, i.e. , such antibodies recognize and bind one Chlamydia peptide and do not substantially recognize or bind to other proteins, including those found in uninfected human cells.
  • the determination that an antibody specifically detects a particular Chlamydia peptide is made by any one of a number of standard immunoassay methods; for instance, the western blotting technique (Sambrook et al. , 1989).
  • total cellular protein is extracted from a sample of blood from an unexposed subject and from a sample of blood from an exposed subject. As a positive control, total cellular protein is also extracted from -21-
  • Chlamydia cells grown in vitro These protein preparations are then electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel. Thereafter, the proteins are transferred to a membrane (for example, nitrocellulose) by western blotting, and the antibody preparation is incubated with the membrane. After washing the membrane to remove non-specifically bound antibodies, the presence of specifically bound antibodies is detected by the use of an anti-guinea pig antibody conjugated to an enzyme such as alkaline phosphatase; application of the substrate 5-bromo-4- chloro-3-indolyl phosphate/nitro blue tetrazolium results in the production of a dense blue compound by immuno-localized alkaline phosphatase.
  • an enzyme such as alkaline phosphatase
  • Antibodies which specifically detect the Chlamydia protein will, by this technique, be shown to bind to the Chlamydia-extmcted sample at a particular protein band (which will be localized at a given position on the gel determined by its molecular weight) and to the proteins extracted from the blood of the exposed subject. No significant binding will be detected to proteins from the unexposed subject.
  • EXAMPLE 4 Use of Infection-Specific Sequences and their Corresponding Peptides and Antibodies in Diagnostic Assays
  • Another aspect of the present invention is a method for detecting the presence of anti- Chlamydia antibodies that react with infection-specific Chlamydia proteins, Chlamydia peptides and Chlamydia nucleic acid sequences in biological samples.
  • These methods include detection of antigen and antibody by ELISA and similar techniques, the detection of proteins in a tissue sample by immunofiuorescence and related techniques and the detection of specific DNA sequences by specific hybridization and amplification.
  • One aspect of the invention is an ELISA that detects anti-Chlamydia antibodies in a medical specimen.
  • An immunostimulatory infection-specific Chlamydia peptide of the present invention is employed as an antigen and is preferably bound to a solid matrix such as a crosslinked dextran such as SEPHADEX (Pharmacia, Piscataway, NJ), agarose, polystyrene, or the wells of a microtiter plate.
  • SEPHADEX Pulpharmacia, Piscataway, NJ
  • the polypeptide is admixed with the specimen, such as blood, and the admixture is incubated for a sufficient time to allow antibodies present in the sample to immunoreact with the polypeptide.
  • the presence of the positive immunoreaction is then determined using an ELISA assay, usually involving the use of an enzyme linked to an anti-immunoglobulin that catalyzes the conversion of a chromogenic substrate.
  • the solid support to which the polypeptide is attached is the wall of a microtiter assay plate.
  • a protein such as bovine serum albumin. Excess bovine serum albumin is removed by rinsing and the medical specimen is admixed with the polypeptide in the microtiter wells. After a sufficient incubation time, the microtiter wells are rinsed to remove excess sample and then a solution of a second antibody, capable of detecting human antibodies is added to the wells.
  • This second antibody is typically linked to an enzyme such as peroxidase, -22-
  • the second antibody may be a peroxidase- labeled goat anti-human antibody. After further incubation, excess amounts of the second antibody are removed by rinsing and a solution containing a substrate for the enzyme label (such as hydrogen peroxide for the peroxidase enzyme) and a color-forming dye precursor, such as o-phenylenediamine is added.
  • a substrate for the enzyme label such as hydrogen peroxide for the peroxidase enzyme
  • a color-forming dye precursor such as o-phenylenediamine
  • Chlamydia peptide bound to the wall of the well
  • human an -Chlamydia antibodies from the specimen
  • enzyme-conjugated anti- human antibody and the color substrate will produce a color that can be read using an instrument that determines optical density, such as a spectrophotometer.
  • optical density such as a spectrophotometer.
  • positive readings indicate the presence of anti-Chlamydia antibodies in the specimen, which in mm indicate a prior exposure of the patient to Chlamydia.
  • antibodies that specifically recognize a Chlamydia peptide encoded by the nucleotide sequences disclosed herein are useful in diagnosing the presence of infection-specific Chlamydia antigens in a subject or sample.
  • detection of infection- specific antigens that are present in cells or tissues may be done by immunofiuorescence, indirect- immunofluorescense and immunohistochemistry.
  • immunofluorescense a fluorescent dye is bound directly to the antibody.
  • indirect-immunofluorescence the dye is bound to an anti- immunoglobulin. Specific binding occurs between antigen and bound antibody is detected by virtue of fluorescent emissions from the dye moiety. This technique may be particularly useful, for instance, for detection of Chlamydia antigen present on a urogenital mucosal smear.
  • Chlamydia may be present in urogenital mucosa, and a smear on a glass slide may be fixed and bathed in a solution containing an antibody specific to the infection-specific antigen. The slide is then washed to remove the unbound antibody, and a fluorescent anti-immunoglobulin antibody is added. The slide is washed again, and viewed microscopically under an appropriate wavelength of light to detect fluorescence. Fluorescence indicates the presence of Chlamydia antigen.
  • a urogenital mucosal smear may be taken, the sample cultured with HeLa cells to produce large amounts of the RB form, and immunofiuorescence may then be used to detect infection-specific Chlamydia antibodies.
  • nucleic acid primers to detect the presence of Chlamydia nucleic acids that encode infection-specific antigens in body samples and thus to diagnose infection.
  • these oligonucleotide primers will comprise at least 15 contiguous nucleotides of a DNA sequence as shown in SEQ ID NOS: 1 , 3, 5, 7, 9, 11, 13, 15, or 17.
  • such oligonucleotides may comprise at least 20 or at least 25 or more contiguous nucleotides of the aforementioned sequences.
  • PCR primers are not required to exactly match the target gene sequence to which they anneal.
  • the oligonucleotides will comprise a sequence of at least 15 nucleotides and preferably at least 20 nucleotides, the oligonucleotide sequence being substantially similar to a DNA sequence set forth -23-
  • Such oligonucleotides may share at least about
  • PCR polymerase chain reaction amplification
  • PCR amplification employing primers based on the sequences disclosed herein may also be employed to quantify the amounts of Chlamydia nucleic acid present in a particular sample (see chapters 8 and 9 of Innis et al. , (1990)).
  • probes based on the nucleic acid sequences described herein may be labeled with suitable labels (such a P 32 or biotin) and used in hybridization assays to detect the presence of
  • Chlamydia nucleic acid in provided samples may also be utilized to detect the presence of Chlamydia RNA which is indicative of an ongoing infection.
  • the purified peptides of the present invention may be used directly as immunogens for vaccination.
  • Methods for using purified peptides as vaccines are well known in the art and are described in Yang et al. (1991), Andersen (1994) and Jardim et al. (1990).
  • adjuvants such as alum, Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IF A) may be used in formulations of purified peptides as vaccines.
  • one embodiment of the present invention is a vaccine comprising one or more immunostimulatory C. trachomatis or C. psittaci peptides encoded by nucleotide sequences as shown in the attached sequence listing, together with a pharmaceutically acceptable adjuvant.
  • a vaccine may comprise a defined fraction of the disclosed peptide of C. trachomatis or C. psittaci or may comprise a peptide wherein the gene coding for the peptide shows substantial similarity to the DNA sequences disclosed herein, such as for orthologous genes of C. pneumoniae or C. pecorum.
  • the vaccines may be formulated using a peptide according to the present invention together with a pharmaceutically acceptable excipient such as water, saline, dextrose and glycerol.
  • a pharmaceutically acceptable excipient such as water, saline, dextrose and glycerol.
  • the vaccines may also include auxiliary substances such as emulsifying agents and pH buffers. It will be appreciated by one of skill in the art that vaccines formulated as described above may be administered in a number of ways including subcutaneous, intra-muscular and intra- venous injection. Doses of the vaccine administered will vary depending on the antigenicity of the particular peptide or peptide combination employed in the vaccine, and characteristics of the -24-
  • the vaccines of the present invention may routinely be administered several times over the course of a number of weeks to ensure that an effective immune response is triggered. Where such multiple doses are administered, they will normally be administered at from two to twelve week intervals, more usually from three to five week intervals.
  • Periodic boosters at intervals of 1-5 years, usually three years, may be desirable to maintain the desired levels of protective immunity.
  • multiple immunostimulatory peptides may also be administered by expressing the nucleic acids encoding the peptides in a nonpathogenic microorganism, and using this transformed nonpathogenic microorganism as a vaccine.
  • nucleic acid molecules encoding peptide antigens as described in Janeway & Travers, (1997).
  • plasmids which include nucleic acid molecules described herein, or which include nucleic acid sequences encoding peptides according to the present invention may be utilized in such DNA vaccination methods.
  • the vaccine of the invention may be used to inoculate potential animal targets of any of the chlamydial diseases including those caused by C. trachomatis, C. psittaci, C. pneumoniae or C. pecorum. Indeed the vaccine of the invention may be used to inoculate animals against any disease that shows immunological cross-protection as a result of exposure to infection-specific
  • Chlamydia antigen The protein or polypeptide is present in the vaccine in an amount sufficient to induce a protective immune response whether through humoral or cell mediated pathways or through both. Such a response protects the immunized animal against chlamydial infections specifically by raising an immune response against the Reticulate Body form of Chlamydia.

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US8206724B2 (en) 2000-12-15 2012-06-26 Auburn University Method and compositions for vaccination comprising nucleic acid and/or polypeptide sequence of chlamydia
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US7374059B2 (en) 2001-10-05 2008-05-20 Industrial And Environmental Concepts, Inc. Covering systems and venting methods
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