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
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/118—Chlamydiaceae, e.g. Chlamydia trachomatis or Chlamydia psittaci
• • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/10—Antimycotics
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
  • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
  • C07K16/125—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Chlamydiales (O)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56911—Bacteria
  • G01N33/56927—Chlamydia
• • 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
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • 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
  • A61K38/00—Medicinal preparations containing peptides
• • 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

• This invention relates to antigenic proteins from Chlamydia trachomatis.
• it relates to antigens which are recognised by antibodies from chronically infected or convalescent patient sera.
• Chlamydia are obligate intracellular parasites of eukaryotic cells which are responsible for endemic sexually transmitted infections, trachoma, infectious pneumonitis, and various other disease syndromes. They occupy an exclusive eubacterial phylogenic branch, having no close relationship to any other known organisms - they are classified in their own order (Chlamydiales) which contains a single family (Ciilamydiaceae) which in turn contains a single genus (Chlamydia).
• Chlamydiales which contains a single family
• Ciilamydiaceae which in turn contains a single genus
• Four chlamydial species are currently known - C trachomatis, C.pneumoniae, C.pecorum and C.psittaci [eg. see reference 1].
• a genome sequence of C. trachomatis has recently been published [2].
• Serovars A-K elicit epithelial infections primarily in the ocular tissue (A-C) or urogenital tract (D-K).
• Serovars LI . L2 and L3 are the agents of invasive lymphogranuloma venereum (LGV).
• chlamydial infection itself causes disease, it is thought that, in some patients, the severity of symptoms is due, in fact, to an aberrant host immune response. Failure to clear the infection results in persistent immune stimulation and, rather than helping the host, this results in chronic infection with severe consequences, including sterility and blindness [3].
• vaccines are useful (a) for immunisation against chlamydial infection or against chlamydia-induced disease (prophylactic vaccination) or (b) for the eradication of an established chronic chlamydial infection (therapeutic vaccination). Being an intracellular parasite, however, the bacterium can generally evade antibody-mediated immune responses.
• the invention is based on the identification of proteins encoded by Chlamydia trachomatis which are immunogenic in man as a consequence of infection.
• the invention provides a Ctrachomatis protein having the MW and pi characteristics of protein 5, 6, 7, 8, 9, 1 1, 13, 14, 15, 16, 17. 18, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42. 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, or 55, as set out in Table II on page 15.
• the invention further provides proteins comprising fragments of the Ctrachomatis proteins of the invention.
• the fragments should comprise at least n consecutive amino acids from the proteins and, depending on the particular protein, n is 7 or more (eg. 8, 10, 12, 14, 16, 18, 20, 50, 100 or more).
• n is 7 or more (eg. 8, 10, 12, 14, 16, 18, 20, 50, 100 or more).
• the fragments comprise an epitope from the protein.
• the proteins of the invention can, of course, be prepared by various means (eg. recombinant expression, purification from cell culture, chemical synthesis etc.) and in various forms (eg. native, fusions etc.). They are preferably prepared in substantially isolated or purified form (ie. substantially free from other Ctrachomatis or host cell proteins)
• the invention provides antibodies which bind to these proteins. These may be polyclonal or monoclonal and may be produced by any suitable means. -
• the invention provides nucleic acid encoding the proteins and protein fragments of the invention. Nucleic acid having sequence identity to this nucleic acid is also provided. Depending on the particular nucleic acid, the degree of identity is preferably greater than 50% (eg. 65%, 80%, 90%. 95%. 98%. 99% or more). Furthermore, the invention provides nucleic acid which can hybridise to this nucleic acid, preferably under "high stringency " conditions (eg. 65°C in a O.lxSSC, 0.5% SDS solution).
• fragments of this nucleic acid are also provided.
• the fragments should comprise at least n consecutive nucleotides from the sequences and, depending on the particular sequence, n is 10 or more (eg. 12, 14, 15, 18, 20, 25, 30, 35. 40 or more). It should also be appreciated that the invention provides nucleic acid comprising sequences complementary to those described above (eg. for antisense or probing purposes).
• Nucleic acid according to the invention can, of course, be prepared in many ways (eg. by chemical synthesis, from genomic or cDNA libraries, from the organism itself etc.) and can take various forms (eg. single stranded, double stranded, vectors, probes etc.).
• nucleic acid includes DN A and RNA, and also their analogues, such as those containing modified backbones, and also peptide nucleic acids (PNA) etc.
• the invention provides vectors comprising nucleic acid of the invention (eg. expression vectors) and host cells transformed with such vectors.
• nucleic acid of the invention eg. expression vectors
• compositions comprising protein, antibody, and/or nucleic acid according to the invention.
• These compositions may be suitable as immunogenic compositions (including vaccines), for instance, or as diagnostic reagents.
• the invention also provides nucleic acid, protein, or antibody according to the invention for use as medicaments (eg. as vaccines) or as diagnostic reagents.
• the invention provides protein 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20. 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37. 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 (as set out in Table II on page 15) for use as a chlamydial immunogen. Whilst it is believed that some of the proteins described in Table II may be known per se, they have not been disclosed as being immunogenic.
• the invention also provides the use of nucleic acid, protein, or antibody according to the invention in the manufacture of (i) a medicament for treating or preventing infection due to
• Chlamydia (ii) a diagnostic reagent for detecting the presence of Chlamydia or of antibodies raised against Chlamydia: and/or (iii) a reagent which can raise antibodies against Chlamydia.
• the Chlamydia may be any species or strain, but is preferably Ctrachomatis.
• the invention provides a protein of the 55 proteins of Table II for use in such manufacture.
• the invention also provides a method of treating a patient, comprising administering to the patient a therapeutically effective amount of nucleic acid, protein, and/or antibody according to the invention.
• the invention provides various processes.
• a process for producing proteins of the invention comprising the step of culturing a host cell according to the invention under conditions which induce protein expression.
• a process for producing protein or nucleic acid of the invention wherein the protein or nucleic acid is synthesised in part or in whole using chemical means.
• a process for detecting nucleic acid of the invention comprising the steps of: (a) contacting a nucleic probe according to the invention with a biological sample under hybridising conditions to form duplexes: and (b) detecting said duplexes.
• a process for detecting proteins of the invention comprising the steps of: (a) contacting an antibody according to the invention with a biological sample under conditions suitable for the formation of an antibody-antigen complexes; and (b) detecting said complexes.
• the invention provides a process for detecting anti-chlamydial antibodies in a sample, comprising the steps of: (a) contacting a protein according to the invention with a biological sample under conditions suitable for the formation of an antibody-antigen complexes; and (b) detecting said complexes.
• kits comprising reagents suitable for use in these processes.
• a kit comprising a nucleic probe according to the invention and means for detecting duplexes formed by the probe.
• a kit is provided comprising an antibody according to the invention and means for detecting antibody-antigen complexes formed by the antibody.
• a kit comprising a protein according to the invention and means for detecting antibody-antigen complexes formed by the protein.
• composition "comprising" X may consist exclusively of X or may include something additional to X, such as X+Y.
• Figure 1 shows the annotated reference 2D electrophoretic EB map, also indicating the positions of the immunoreactive protein spots, labelled 1-55. Groups of spots which appear to be an isolectric series of the same protein are encircled together and classified under the same identification number.
• Figure 2 shows typical immunoblots. The whole map area is shown. Major known immunogens are marked for easier comparison. For other spot identification, refer to Figure 1 and table II.
• Blot A is from PID patient .1051 (MIF titre 256), and has a serum dilution 1 :5000.
• Blot B is from patient JO35 (MIF titre 64) affected by secondary sterility, and has a serum dilution 1 :2500.
• Blot C is similar to blot B, but is from patient O52.
• Blot D is from PID patient JO31 (MIF titre 256), and gas a serum dilution 1 :5000.
• Sera (Table I) were obtained from women who had responded to a chlamydial infection of the genital tract.
• the seventeen sera (A...Q) were obtained from 4 cases of lower genital tract infection and 13 laparoscopically-confirmed cases of PID (pelvic inflammatory disease), including 2 cases of secondary sterility. All sera were positive for a standard microimmunofluorescence test (MIF) with purified Ctrachomatis L2 elementary bodies [1 1], and confirmed as Ctrachomatis immune sera by an ELISA test with the plasmid-encoded pgp3 antigen [5].
• MIF microimmunofluorescence test
• a group of 10 seronegative control sera from healthy blood donors was tested by immunoblotting in the same way. and using the same dilutions as for patient sera, in order to exclude the occurrence of non-specific reactions.
• Chlamydial proteins were separated using high resolution 2D electrophoresis, performed using the immobiline/polyacrylamide system, essentially as described in references 14 and 15.
• the second dimension was carried out in a Laemmli system on 9-16% polyacrylamide linear gradient gels (18 cm x 20 cm x 1.5 mm), at 40 mA/gel constant current, for approximately 5 hours until the dye front reached the bottom of the gel.
• Analytical gels were stained with ammoniacal silver nitrate [16].
• the protein maps were scanned with a laser photodensitometer (Molecular Dynamics) and converted into electronic files which were then analysed with the Melanie II computer software (Bio-Rad).
• Figure 1 shows the annotated reference EB map which was used to identify proteins on immunoblots. MW and pi coordinates for the reference map were calibrated by co-migration of the chlamydial proteins with human serum proteins acting as reference proteins. The isoelectric point values used for serum proteins were those described in reference 17.
• the gels were electroblotted onto nitrocellulose membranes [18], and processed according to standard procedures, modified as described in reference 19. Briefly, before immunodetection. the membranes were stained in 0.2% (w/v) Ponceau S in 3% (w/v) trichloroacetic acid for 3 minutes and the positions of selected anchor spots were marked on the blot to assist matching of the immunoblots with the silver stained map. Immunoreactive spots were detected by overnight incubation at room temperature with patient sera (1500-5000x dilutions), followed by incubation with rabbit anti-human IgGs conjugated with peroxidase (Cappel. 7000x dilution), and detection with a chemiluminescence based kit (Pharmacia Amersham Biotech).
• the nitrocellulose blots were marked with a number of internal "anchor" spots using transient Ponceau Red staining. After incubation with the sera and detection of bound antibodies by chemiluminescence, the immunoblot images were matched to the reference map and spots were assigned the corresponding pi and MW coordinates (see Table II). When the position and shape of the spot (or isoelectric series of spots) coincided with a previously-identified EB antigen, an immune response against such antigen was recorded. In all other cases the immunoblot spot was identified by the MW and pi coordinates taken at the baricentre of the stained area (or the coordinate range, in the case of complex spot patterns). It will be appreciated that the MW and pi values are determined electrophoretically, and may have a potential average error of +/-10%. The higher MW measurements will tend to be less accurate.
• This cluster is shown as spot 1 in Figure 1 - all the spots labelled "1 *" were scored as a single antigen, but a number of accessory spots with lower MW and pi values which usually appear associated to OMP2 reactivity were separately scored, as their relationship to the OMP2 polypeptide is still unclear. Because the OMP2 protein is chlamydia-specific. and does not seem to undergo any relevant antigenic variation, it can be considered probably the best marker of chlamydial infection in this study.
• 2D maps were prepared as described above, starting from lmg total EB protein per run, followed by blotting onto polyvinylidene difluoride membranes (BioRad PVDF membranes 20 x 20 cm, 0.2 micron pore size), as in reference 20.
• the blots were stained with 0.1% (w/v) Coomassie Brilliant Blue R250 in 50% aqueous methanol for 5 minutes, and de-stained in 40% methanol, 10% acetic acid.
• Membranes were dried at 37°C and stored at -20°C for further analysis.
• Selected protein spots were cut out and submitted to amino acid sequencing by Edman degradation using an automatic Protein/Peptide Sequencer (mod 470A; Applied Biosystem Inc.) connected on-line with a phenylthiohydantoin-amino acid analyser model 120A and a control/Data Module model 900A (Applied Biosystems Inc.). Typically 3 or 4 equivalent spots from similar blots were used, according to the estimated relative molar amount of protein in the spot.
• Spot 38 predicted to be a GTP-binding protein (currently annotated as ychF).
• the N-terminal sequences of spots 26, 31 and 33 do not match any database sequences, including the published serovar D sequence.
• Table IV shows a summary of identifications (some putative) of several immunoreactive antigens, which were obtained either by comparison with previous 2D mapping data, or by homology searches with the N-terminal sequencing data obtained above.
• This spot is believed to be the alpha chain of the Ctrachomatis RNA polymerase (gi620029), based on its MW/pI position, and on its N-terminus sequence.
• RNAP alpha chain has previously been described [23], it has never been reported as a chlamydial immunogen.
• Four patients showed reactivity to this protein, demonstrating that it is immunogenic in humans as a consequence of chlamydial infection.
• the intracellular parasitic nature of Chlamydia means that it can generally evade antibody-mediated immune responses
• the antibody reactivity demonstrated above indicates that the immune system does encounter these proteins during natural infection, and the formation of antibodies may, for instance, also help to prime the T-cell-mediated immune responses.
• Spots 18 and 46 appear to be homologous to the ompB gene in the serotype D genome, annotated as encoding a putative outer membrane protein.
• the N-terminal sequences and pi & MW values are in agreement with the expected properties of an ompB gene product, after cleavage of the predicted N-terminal signal peptide.
• spots 18 and 46 are present in a 2D electrophoretic map of a purified preparation of chlamydial outer membrane complex, which also supports the view that spots 18 and 46 represent the homologs of the serotype D ompB gene.
• This protein appears as two distinct electrophoretic species, but a spot shift of this type is usually associated to a variation of amino acid composition, either due to amino acid sequence variation, and/or to true or artefactual derivatisation of some amino acid residues. Five patients showed reactivity to this protein, demonstrating that it is immunogenic in humans as a consequence of chlamydial infection.
• This spot is believed to be an aminopeptidase. based on its MW/pI position and on its N-terminus sequence (both in comparison with the published serovar D sequence pepA). Four patients showed reactivity to this protein, demonstrating that it is immunogenic in humans as a consequence of chlamydial infection.
• This spot is believed to be a GTP -binding protein, based on its MW/pI position and on its N-terminus sequence (both in comparison with the published serovar D sequence ychF). Two patients showed reactivity to this protein, demonstrating that it is immunogenic in humans as a consequence of chlamydial infection.
• This spot is believed to be a stress-induced protease, based on its MW/pI position and on its N-terminus sequence (both in comparison with the published serovar D sequence htrA). Seven patients showed reactivity to this protein, demonstrating that it is immunogenic in humans as a consequence of chlamydial infection.
• Cys and Tip are not determined in this type of analysis, and it is not possible to distinguish between Glu/Gln and Asp/Asn.
• N-terminal residue is blocked due to some form of modification (eg. a lipoprotein).
• Modification is often a characteristic of membrane-associated proteins in eukaryotes, but is also a characteristic of outer surface proteins or secreted proteins in bacterial species (eg. lipoproteins [24], mycoplasma outer membrane proteins [25], the FHA virulence factor of B.pertussis [26] etc.).
• the letters in the first column correspond to those given in Table II.
• the codes in the second column refer to the original serum collection.
• the pathology associated with each patient is broadly indicated as cervicitis (lower genital tract infection), PID or sterility (secondary to infection). All sera were characterised by MIF assay with purified L2 elementary bodies.
• the MIF titre given in the table is the highest two-fold dilution which gave a positive signal.
• the 'Best Dilution' is the dilution which was found to give minimum background without loss of signal on weaker spots.
• CT-D gene refers to the gene name from reference 2 and gives the names of genes likely to encode homologue proteins in Ctrachomatis D. Theoretical pI/MW values in the last column, to be compared to the experimental values, were calculated the from CT-D gene sequences.

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