WO2007030879A1 - Marqueurs diagnostiques et leurs utilisations - Google Patents

Marqueurs diagnostiques et leurs utilisations Download PDF

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Publication number
WO2007030879A1
WO2007030879A1 PCT/AU2006/001346 AU2006001346W WO2007030879A1 WO 2007030879 A1 WO2007030879 A1 WO 2007030879A1 AU 2006001346 W AU2006001346 W AU 2006001346W WO 2007030879 A1 WO2007030879 A1 WO 2007030879A1
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proteins
infection
seq
subject
acute
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PCT/AU2006/001346
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English (en)
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Peter Timms
Trudi Anne Armitage
Thomas Bernard Macnaughton
Terence Patrick Walsh
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Diatech Pty Ltd
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Priority claimed from AU2005905048A external-priority patent/AU2005905048A0/en
Application filed by Diatech Pty Ltd filed Critical Diatech Pty Ltd
Publication of WO2007030879A1 publication Critical patent/WO2007030879A1/fr

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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • the present invention relates generally to methods for diagnosing and treating infectious diseases and other conditions related thereto. More particularly, the present invention relates to methods for determining the presence of organisms of the Chlamydiaceae family in a subject, including species of Chlamydia, and to methods for determining the stage of an infection caused by such organisms.
  • the present invention also relates to kits for use with the diagnostic methods. The methods and kits of the present invention are particularly useful in relation to human and non-human, i.e. veterinary subjects.
  • the present invention further relates to methods for identifying proteins or nucleic acid sequences associated with chlamydial infection in a subject.
  • proteins or nucleic acid sequences are not only useful in relation to the diagnostic methods of the invention but are also useful in the development of methods and agents for preventing and/or treating chlamydial infection in a subject, such as but not limited to, immunotherapeutic methods and agents.
  • Organisms of the Chlamydiaceae family are an important class of human pathogens. Chlamydia trachomatis, for example, is the most common sexually transmitted pathogen with an estimated 3 million new infections reported annually in the United States alone (Groseclose et al, Sex Transm Dis 2(5:339-344, 1996). This distinct pathogen has the ability to exist in acute, chronic or persistent states of infection. An untreated C.
  • trachomatis infection can lead to various disease states such as salpingitis (fallopian tube inflammation), pelvic inflammatory disease (PID), tubal occlusion and infertility (Cates et al, Am J Obstet Gynecol 164:1771-81, 1991; Cohen and Brunham, Sex Transm Infect 75:21-24, 1999).
  • salpingitis fallopian tube inflammation
  • pelvic inflammatory disease PID
  • tubal occlusion and infertility Cates et al, Am J Obstet Gynecol 164:1771-81, 1991; Cohen and Brunham, Sex Transm Infect 75:21-24, 1999.
  • PCR nucleic acid amplification testing
  • EIA direct fluorescent antibody and enzyme immunoassay
  • MIF microimmunofluorescence
  • sequence identifier number Nucleotide and amino acid sequences are referred to by sequence identifier number (SEQ ID NO:).
  • the SEQ ID NOs: correspond numerically to the sequence identifiers ⁇ 400>l (SEQ ID NO:1), ⁇ 400>2 (SEQ ID NO:2), etc.
  • a summary of the sequence identifiers is provided in Table 1.
  • a sequence listing is provided at the end of the specification.
  • the present invention is predicated in part by the identification of a differential antibody response to specific antigens in the sera of subjects infected with organisms of the Chlamydiaceae family, and in particular, species of Chlamydia.
  • This identification allows not only the presence of a chlamydial infection to be determined in a subject, but also the determination of the stage of the chlaymidial infection in a subject if it is determined to be present.
  • the identification of immunoreactive proteins also enables the development of vaccines and other therapeutic protocols for the treatment or prevention of chlamydial infection.
  • the present invention provides a method for determining the presence or stage of chlamydial infection in a subject, said method comprising detecting the presence and/or amount of one or more proteins or variants thereof which are associated with chlamydial infection in a subject, or one or more expressed nucleic acid sequences or variants thereof encoding said proteins, or one or more antigen-binding molecules specific for said proteins, wherein the presence and/or amount of said proteins, nucleic acid sequences or antigen-binding molecules indicates the presence or stage of chlamydial infection in a subject.
  • the method of the present invention may be performed by detecting proteins, nucleic acid sequences, or antigen-binding molecules, preferably antigen-binding molecules are detected.
  • the antigen-binding molecules are derived from a subject and are specific for proteins associated with chlamydial infection in said subject, such as chlamydial antigens.
  • the present invention provides a method for determining the presence or stage of chlamydial infection in a subject, said method comprising:
  • the antigen-binding molecules are preferably antibodies.
  • the antibodies may be specific for antigens from a strain of Chlamydia which predominately infects female subjects or male subjects or both female and male subjects.
  • preferred antigens include proteins designated CT314 (DNA-directed RNA polymerase), CT147 (protein), CT727 (metal transport P-type ATPase), CT396 (heat shock protein 70), CTl 57 (phospholipase D endonuclease), CT423 (hemolysin-like protein) and CT413 (probable outer membrane protein) as well as homologs or variants thereof.
  • CT314 DNA-directed RNA polymerase
  • CT147 protein
  • CT727 metal transport P-type ATPase
  • CT396 heat shock protein 70
  • CTl 57 phospholipase D endonuclease
  • CT423 hemolysin-like protein
  • CT413 probable outer membrane protein
  • the present invention provides a method for determining the presence or stage of chlamydial infection in a subject, said method comprising detecting the presence and/or amount of antibodies in a biological sample from said subject to one or more proteins selected from the list consiting of CT147 (SEQ ID NO:2), CT314 (SEQ ID NO:4), CT727
  • CT413 SEQ ID NO: 13 wherein the presence of antibodies to one or more of said protein indicates the presence or stage of chlamydial infection in said subject.
  • the present invention contemplates a method for determining the presence or stage of chlamydial infection in a subject, said method comprising detecting the presence and/or amount of antibodies in a biological sample from said subject to one or more proteins selected from the list consisting of CT147 (SEQ ID NO:2), CT314 (SEQ ID NO:4), CT727 (SEQ ID NO:6), CT396 (SEQ ID NO:8), CT423 (SEQ ID NO: 10), CTl 57 (SEQ ID NO:12) and CT413 (SEQ ID NO:13) wherein:
  • Reference to "a greater level of antibodies” refers to a higher trite in a person infected with Chlamydia compared to a subject never previously disposed to Chlamydia. It includes greater amounts of from approximately 0.5% to 100% such as 0.5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.
  • the present invention also provides a kit suitable for use with a method for determining the presence or stage of chlamydial infection in a subject, said kit comprising components which facilitate the detection of the presence and/or amount of one or more proteins or variants thereof which are associated with chlamydial infection in a subject, or one or more nucleic acid sequences or variants thereof encoding said proteins, one or more antigen- binding molecules specific for said proteins.
  • the kit of the present invention may be used to detect proteins, nucleic acid sequences or antigen-binding molecules, preferably antigen-binding molecules derived from a subject, such as antibodies, are detected.
  • the kit of the present invention preferably comprises one or more chlamydial antigens.
  • the chlamydial antigens may be provided immobilised on a solid substrate, or alternatively, the chlamydial antigens may be provided free in solution.
  • the kit may also be in the form of a panel of antigens.
  • the antigens may be purified naturally occurring molecules or recombinant molecules, fusion molecules or an antibody-binding fragment of the antigen.
  • the present invention provides a kit for identifying a chlamydial infection or for distinguishing between stages of a chlamydial infection, said kit comprising a support or container adapted to contain one or more proteins selected from CTl 47, CT314, CT727, CT396, CT423, CTl 57 and CT413, said support of container capable of receiving a biological sample potentially comprising antibodies to one or more of said proteins.
  • the present invention also provides a method for identifying proteins, nucleic acid sequences and antigen-binding molecules associated with chlamydial infection in a subject which are suitable for use with the diagnostic methods and kits described herein. Such proteins or nucleic acid sequences are also useful in the development of methods and agents for preventing and/or treating chlamydial infection in a subject, such as, but not limited to, immunotherapeutic methods and agents.
  • the present invention provides a method for preventing and/or treating chlamydial infection in a subject said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient to prevent and/or treat chlamydial infection in said subject.
  • the present invention contemplates a method for vaccinating a subject against chlamydial infection said method comprising administering to said subject an antibody- inducing effective amount of one or more proteins selected from CT147, CT314, CT727, CT396, CT423, CT157 and CT413 or an immunogenic fragment thereof.
  • the present invention further provides a vaccine against chalymidal infection said vaccine comprising at least one protein selected from CT147, CT314, CT727, CT396, CT423, CT157 and CT413 or an antigenic fragment thereof said vaccine further comprising one or more pharmaceutically acceptable carriers, diluents, excipients, adjuvants and/or immune response enhancers.
  • the agent is an immunotherapeutic agent that is in the form of an immunotherapeutic composition such as, but not limited to, a vaccine.
  • the present invention also provides for the use of the agent for the preparation of a composition.
  • the vaccine may comprise one or more of CT314, CT147, CT727, CT396, CTl 57, CT423 and/or CT413 and optionally an adjuvant or immune potentiating agent or pharmaceutically acceptable carrier or excipient.
  • kits for identifying a chlamydial infection or for distinguishing between strains of Chlamydia comprising a support or container adapted to contain one or more proteins selected from CT147, CT314, CT727, CT396, CT423, CT157 and CT413 said proteins, said support or contain capable of receiving a biological sample potentially comprising antibodies to one or more of said proteins.
  • the kit may contain one or two or three of four or five or six or all seven CT proteins.
  • the present invention further provides an isolated protein selected from the list consisting of CT147, CT314, CT727, CT396, CT423, CT157 and CT413.
  • the methods, kits and agents contemplated by the present invention may be used in relation to any infection caused by organisms of the Chlamydiaceae family.
  • the infection is caused by Chlamydia trachomatis.
  • the methods, kits and agents contemplated by the present invention are also useful in relation to conditions that are related to, or otherwise arise from, chlamydial infection such as a disease of the systemic vasculature (e.g. heart and lung disease).
  • Figure 1 is a photographic representation showing Western blots of uninfected (UI) and infected (I) whole cell extracts probed with sera from 5 patient groups: (a) Acute, (b) Recovered Acute, (c) PID, (d) Infertile Control and (e) Negative Control. Circled are the four identified differential chlamydial antigenic bands designated A, B, C and D.
  • Figure 2 is a photographic representation showing sera from five patient groups (a) Acute, (b) Recovered Acute, (c) PID and (d) negative control were probed against C. trachomatis L2, D and K and C. pneumoniae to determine the potential species and serovar specificity of bands A, B, C and D.
  • Figure 3 is a graphical representation showing that in the acute phase of infection, ATPase reactivity is low compared to CTl 47, Hemolysin and 13.5kDa which demonstrate moderate levels of antigenic reactivity.
  • all antigens show a higher reactivity with ATPase displaying the greatest increase.
  • the chronic (PID) stage antibody responses to CT 147 are reduced, and an equal reactivity of ATPase and
  • Figure 4 is a photographic representation showing western blot of uninfection (UI) and infected (I) whole cell extracts probed with sera from seven patient groups: (a) Acute, (b) Recovered Acute, (c) 204, (d) 212 (e) Chronic, (f) Adult Male Control and (g) Child Male Control. Circled are three of the identified differential antigenic bands (designated B, C and D) previously witnessed in the various femal patient groups. Boxed is the novel male marker designated M.
  • Figure 7 is a graphical representing showing high levels of antigen reactivity seen in the acute phase of infection.
  • the recovery phase of infection shows an overall decrease in antibody production compared to the 204 group where reactivity to all four bands is dramatically increased.
  • a reduction in antibody production to bands B, C and D, and a total absence of reactivity to bands D and M is observed for 212 patients. Only reactivity to bands B and C are observed in the chronic group.
  • an "antigen-binding molecule” includes a single antigen-binding molecule, as well as two or more antigen-binding molecules; reference to an “antigen” includes a single antigen, as well as two or more antigens; and so forth.
  • the present invention provides a method for determining the presence or stage of chlamydial infection in a subject, said method comprising detecting the presence and/or amount of one or more proteins or variants thereof which are associated with chlamydial infection in a subject, or one or more nucleic acid sequences or variants thereof encoding said proteins, or one or more antigen-binding molecules specific for said proteins, wherein the presence and/or amount of said proteins, nucleic acid sequences or antigen-binding molecules indicates the presence or stage of chlamydial infection in a subject.
  • Chlamydial infection refers to the establishment and growth of a population of chlamydial organisms in a subject. Chlamydial infection can result in a number of pathological conditions such as infertility, salpingitis, tubal occlusion pelvic inflammatory disease (PID), urethritis, epididymitis, proctitis, conjunctivitis, dysuria, trachoma and psittacosis. Some forms of chlamydial infection can also exist in a subject in different "stages", such as acute and chronic stages.
  • stages such as acute and chronic stages.
  • Subject as used herein refers to humans and non-human primates (e.g. gorilla, macaque, marmoset), livestock animals (e.g. sheep, cow, horse, donkey, pig, chicken), companion animals (e.g. dog, cat, parrot), laboratory test animals (e.g. mouse, rabbit, rat, guinea pig, hamster) and any other organisms which can benefit from the methods, kits and agents of the present invention.
  • the most preferred subject of the present invention is a human.
  • the diagnostic method of the present invention may be performed by detecting either proteins or variants thereof which are associated with chlamydial infection in a subject, or the nucleic acid sequences or variants thereof encoding said proteins or antigen-binding molecules specific for said proteins.
  • Reference herein to "associated with chlamydial infection” means that the presence or amount of the proteins, nucleic acids or antigen- binding molecules is dependent upon, or is otherwise regulated by, the presence or stage of a chlamydial infection.
  • the proteins associated with chlamydial infection in a subject are selected from the group comprising CT147 [protein] (SEQ ID NO:2) 3 CT314 [DNA-directed RNA polymerase] (SEQ ID NO:4), CT727 [metal transport P-type ATPase] (SEQ ID NO:6), CT396 [heat shock protein 70] (SEQ ID NO:8), CT423 [hemolysin-like protein] (SEQ ID NO: 10), CTl 57 [phospholipase D endonuclease] (SEQ ID NO: 12) and CT413 (SEQ ID NO: 13).
  • CT147 [protein] SEQ ID NO:2) 3 CT314 [DNA-directed RNA polymerase] (SEQ ID NO:4)
  • CT727 metal transport P-type ATPase]
  • CT396 heat shock protein 70]
  • CT423 [hemolysin-like protein]
  • SEQ ID NO: 10 CTl 57 [phospholipase D endon
  • the present invention comprises detecting the presence and/or amount of one or more nucleic acid sequences or variants thereof encoding one or more proteins which are associated with chlamydial infection in a subject, wherein the presence and/or amount of said nucleic acid sequences indicates the presence or stage of chlamydial infection in a subject.
  • the present invention provides a method for determining the presence or stage of chlamydial infection in a subject, said method comprising detecting the presence and/or amount of antibodies in a biological sample from said subject to one or more proteins selected from the list consisting of CT147 (SEQ ID NO:2), CT314 (SEQ ID NO:4). CT727 (SEQ ID NO:6), CT396 (SEQ ID NO:8), CT423 (SEQ ID NO: 10), CTl 57 (SEQ ID NO : 12) and CT413 (SEQ ID NO : 13) wherein the presence of antibdoies to one or more of said protein indicates the presence or stage of chlamydial infection in said subject.
  • CT147 SEQ ID NO:2
  • CT314 SEQ ID NO:4
  • CT727 SEQ ID NO:6
  • CT396 SEQ ID NO:8
  • CT423 SEQ ID NO: 10
  • CTl 57 SEQ ID NO : 12
  • CT413 SEQ ID NO : 13
  • the present invention contemplates a method for determining the presence or stage of chlamydial infection in a subject, said method comprising detecting the presence and/or amount of antibodies in a biological sample from said subject to one or more proteins selected from the list consisting of CT147 (SEQ ID NO:2), CT314 (SEQ ID NO:4), CT727 (SEQ ID NO:6), CT396 (SEQ ID NO:8), CT423 (SEQ ID NO: 10), CTl 57 (SEQ ID NO:12) and CT413 (SEQ ID NO:13) wherein:
  • Reference to "a greater level of antibodies” refers to a higher trite in a person infected with Chlamydia compared to a subject never previously disposed to Chlamydia. It includes greater amounts of from approximately 0.5% to 100% such as 0.5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.
  • nucleic acid sequence may be used interchangeably with “oligonucleotide” and “polynucleotide” and as used herein refers to DNA, cDNA, RNA, mRNA or cRNA. Nucleic acid sequences can be isolated from cells contained in a biological sample, according to standard methodologies (Sambrook et al, "Molecular Cloning. A Laboratory Manual”, Cold Spring Harbor Press, 1989; Ausubel et al, “Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998).
  • biological sample refers to a sample that may be directly obtained or derived from a subject.
  • the biological sample may be selected from the group consisting of whole blood, serum, a secretion, plasma, saliva, urine, sweat, ascitic fluid, peritoneal fluid, synovial fluid, amniotic fluid, cerebrospinal fluid, tissue biopsy, and the like.
  • the biological sample is selected from a mucosal swab, a throat swab, a urogenital tract swab, an ocular swab, a sputum sample, an aspirate, a nasopharyngeal aspirate, bronchio-alveolar lavage fluids and blood, including whole blood, serum and plasma.
  • the nucleic acid sequences isolated from a cell may be genomic DNA or RNA. Where RNA is isolated, it may be desirable to convert the RNA to a cDNA.
  • the nucleic acid sequences encoding proteins associated with chlamydial infection in a subject are selected from the group comprising CT147 (SEQ ID NO: I) 5 CT314 (SEQ ID NO:3), CT727 (SEQ ID NO:5), CT396 (SEQ ID NO:7), CT423 (SEQ ID NO:9), CT157 (SEQ ID NO:11) and CT413 (SEQ ID NO:13).
  • nucleic acid sequence variants may be deduced from other species belonging to the family Chlamydiaceae by standard protocols known in the art.
  • Nucleic acid sequence variants according to the present invention comprise regions that show at least 50%, preferably at least 55%, more preferably at least 60%, even more preferably at least 65%, even more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90% and still even more preferably at least 95% sequence identity over a reference nucleic acid sequence of identical size ("comparison window") or when compared to an aligned sequence in which the alignment is performed by a computer homology program known in the art.
  • the reference nucleic acid sequence are selected from the group comprising CT147 (SEQ ID NO:1), CT314 (SEQ ID NO:3), CT727 (SEQ ID NO:5), CT396 (SEQ ID NO:7), CT423 (SEQ ID NO:9) and CTl 57 (SEQ ID NO:11).
  • sequence identity means “sequence identity”, “percentage of sequence identity” and “substantial identity”.
  • reference sequence is at least 5 but frequently 10 to 15 and often at least 20 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two nucleic acid sequences may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two nucleic acid sequences, and (2) a sequence that is divergent between the two nucleic acid sequences, sequence comparisons between two (or more) nucleic acid sequences are typically performed by comparing sequences of the two nucleic acid sequences over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window” refers to a conceptual segment of at least 5 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • the comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
  • GAP Garnier et al
  • BESTFIT Pearson FASTA
  • FASTA Altschul et al
  • FASTA Altschul et al
  • TFASTA Pearson's Alignment of Altschul et al
  • Altschul et al Nucl Acids Res 25:3389-3402, 1997.
  • a detailed discussion of sequence analysis can be found in Ausubel et al, "Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998.
  • sequence identity refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
  • a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, GIy, VaI, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, GIu, Asn, GIn, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A,
  • sequence identity will be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software.
  • nucleic acid sequence variants that are substantially complementary to a reference nucleic acid sequence are identified by blotting techniques that include a step whereby nucleic acids are immobilised on a matrix (preferably a synthetic membrane such as nitrocellulose), followed by a hybridisation step, and a detection step.
  • Southern blotting is used to identify a complementary DNA sequence
  • northern blotting is used to identify a complementary RNA sequence.
  • Dot blotting and slot blotting can be used to identify complementary DNA/DNA, DNA/RNA or RNA/RNA polynucleotide sequences.
  • Such techniques are well known by those skilled in the art, and have been described in Ausubel et al, "Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998.
  • complementarity refers to sequences of nucleotides related by the base-pairing rules.
  • sequence “A-G-T-C” is complementary to the sequence “T-C-A-G”.
  • Complementarity may be "partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules.
  • nucleic acids there may be “complete” or “total” complementarity between the nucleic acids.
  • the degree of complementarity between nucleic acids has significant effects on the efficiency and strength of hybridization between nucleic acids.
  • Hybridization as used herein to denote the pairing of complementary nucleotide sequences to produce a DNA-DNA, DNA-RNA or a DNA-PNA hybrid.
  • Complementary base sequences are those sequences that are related by the base-pairing rules. In relation to DNA, A pairs with T and C pairs with G. In relation to RNA U pairs with A and C pairs with G.
  • the base inosine (I) may also be used. Inosine can form base pairs with C or A or G or T (in descending order of stability).
  • the terms "match” and “mismatch” as used herein refer to the hybridization potential of paired nucleotides in complementary nucleic acid strands. Matched nucleotides hybridize efficiently, such as the classical A-T and G-C base pair mentioned above. Mismatches are other combinations of nucleotides that do not hybridize efficiently.
  • Southern blotting involves separating
  • DNA molecules according to size by gel electrophoresis, transferring the size-separated DNA to a synthetic membrane, and hybridising the membrane-bound DNA to a complementary nucleotide sequence labeled radioactively, enzymatically or fluorochromatically.
  • DNA samples are directly applied to a synthetic membrane prior to hybridisation as above.
  • An alternative blotting step is used when identifying complementary polynucleotides in a cDNA or genomic DNA library, such as through the process of plaque or colony hybridisation.
  • a typical example of this procedure is described in Sambrook et al, "Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Press, 1989.
  • Nucleic acid sequences are blotted/transferred to a synthetic membrane, as described above.
  • a reference nucleic acid sequence such as a nucleic acid sequence of the present invention, CT147 (SEQ ID NO:1), CT314 (SEQ ID NO:3), CT727 (SEQ ID NO:
  • CT396 SEQ ID NO:7
  • CT423 SEQ ID NO:9
  • CT157 SEQ ID NO:11
  • polynucleotide variants according to the invention will hybridise to a reference nucleic acid sequence under at least low stringency conditions.
  • Reference herein to low stringency conditions include and encompass from least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridisation at 42° C, and at least about 1 M to at least about 2 M salt for washing at 42° C.
  • Low stringency conditions also may include 1% w/v Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHP04 (pH 7.2), 7% v/v SDS for hybridisation at 65° C 5 and (i) 2xSSC, 0.1% w/v SDS; or (ii) 0.5% w/v BSA, 1 mM EDTA, 40 mM NaHPO 4 (pH 7.2), 5% w/v SDS for washing at room temperature.
  • BSA Bovine Serum Albumin
  • the nucleic acid variants hybridise to a reference polynucleotide under at least medium stringency conditions.
  • Medium stringency conditions include and encompass from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridisation at 42° C, and at least about 0.1 M to at least about 0.2 M salt for washing at 55° C.
  • Medium stringency conditions also may include 1% BSA 5 1 mM EDTA, 0.5 M NaHPO 4 (pH 7.2), 7% w/v SDS for hybridisation at 65° C, and (i) 2 x SSC, 0.1% w/v SDS; or (ii) 0.5% w/v BSA 5 1 mM EDTA 5 40 mM NaHPO 4 (pH 7.2), 5% w/v SDS for washing at 60-65° C
  • the nucleic acid sequence variants hybridise to a reference nucleic acid sequence under high stringency conditions.
  • High stringency conditions include and encompass from at least about 31% v/v to at least about 50% v/v formamide and from about 0.01 M to about 0.15 M salt for hybridisation at 42° C 5 and about 0.01 M to about 0.02 M salt for washing at 55° C.
  • High stringency conditions also may include 1% w/v BSA, 1 mM EDTA 5 0.5 M NaHPO 4 (pH 7.2), 7% w/v SDS for hybridisation at 65° C 5 and (i) 0.2 x SSC, 0.1% w/v SDS; or (ii) 0.5% w/v BSA, ImM EDTA, 40 mM NaHPO 4 (pH 7.2), 1% SDS for washing at a temperature in excess of 65° C.
  • stringent conditions refers to temperature and ionic conditions under which only nucleotide sequences having a high frequency of complementary bases will hybridise. The stringency required is nucleotide sequence dependent and depends upon the various components present during hybridisation. Generally, stringent conditions are selected to be about 10 to 20° C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH. The T m is the temperature (under defined ionic strength and pH) at which 50% of a target sequence hybridises to a complementary probe.
  • T m of a perfectly matched duplex of DNA may be predicted by the formula:
  • T m 81.5 + 16.6 (logic M) + 0.41 (% G+C)-0. 63 (% formamide)- (600/length)
  • M is the concentration of Na + , preferably in the range of 0.01 molar to 0.4 molar;
  • % G+C is the sum of guanosine and cytosine bases as a percentage of the total number of bases, within the range between 30% and 75% G+C;
  • the T m of a duplex DNA decreases by approximately 1° C with every increase of 1% in the number of randomly mismatched base pairs. Washing is generally carried out at T m -15° C for high stringency, or T m -30° C for moderate stringency.
  • a membrane e.g., a nitrocellulose membrane or a nylon membrane
  • immobilised DNA is hybridised overnight at 42° C in a hybridisation buffer (50% v/v deionised formamide, 5xSSC, 5x Denhardt's solution (0.1% v/v ficoll, 0.1% polyvinylpyrollidone and 0.1% bovine serum albumin), 0.1% SDS and 200 mg/niL denatured salmon sperm DNA) containing labeled probe.
  • a hybridisation buffer 50% v/v deionised formamide, 5xSSC, 5x Denhardt's solution (0.1% v/v ficoll, 0.1% polyvinylpyrollidone and 0.1% bovine serum albumin), 0.1% SDS and 200 mg/niL denatured salmon sperm DNA
  • the membrane is then subjected to two sequential medium stringency washes (i.e., 2xSSC, 0.1% w/v SDS for 15 min at 45° C, followed by 2xSSC, 0.1% w/v SDS for 15 min at 50° C), followed by two sequential higher stringency washes (i.e., 0.2xSSC, 0.1% w/v SDS for 12 min at 55° C followed by 0.2xSSC and 0.1% w/v SDS solution for 12 min at 65-68° C.
  • 2xSSC 0.1% w/v SDS for 15 min at 45° C
  • 2xSSC 0.1% w/v SDS for 15 min at 50° C
  • two sequential higher stringency washes i.e., 0.2xSSC, 0.1% w/v SDS for 12 min at 55° C followed by 0.2xSSC and 0.1% w/v SDS solution for 12 min at 65-68° C.
  • Methods for visualising a labeled nucleic acid sequence hybridised to an immobilised nucleic acid sequence are well known to practitioners in the art. Such methods include autoradiography, phosphorimaging, and chemiluminescent, fluorescent and colorimetric detection.
  • Detecting the presence and/or amount of the nucleic acid sequences of the present invention may be performed by any suitable method known to a person skilled in the art. This may include Southern or Northern blotting techniques and may also involve amplification of the nucleic acid sequences.
  • the term "amplification" in this context refers to a biochemical reaction that produces many nucleic copies of a particular target nucleic acid sequence.
  • the reaction is a polymerase chain reaction (PCR) or a similar reaction that uses a polymerase to copy a nucleic acid sequence such as helicase dependent amplification (HDA), transcription mediated amplification (TMA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA) and reverse transcription polymerase chain reaction (RT-PCR).
  • PCR polymerase chain reaction
  • HDA helicase dependent amplification
  • TMA transcription mediated amplification
  • SDA strand displacement amplification
  • NASBA nucleic acid sequence-based amplification
  • RCA rolling circle amplification
  • RT-PCR reverse transcription polymerase chain reaction
  • primer an oligonucleotide which, when paired with a strand of DNA, is capable of initiating the synthesis of a primer extension product in the presence of a suitable polymerising agent.
  • the primer is preferably single-stranded for maximum efficiency in amplification but may alternatively be double-stranded.
  • a primer must be sufficiently long to prime the synthesis of extension products in the presence of the polymerisation agent. The length of the primer depends on many factors, including application, temperature to be employed, template reaction conditions, other reagents, and source of primers. For example, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15 to 35 or more nucleotides, although it may contain fewer nucleotides.
  • Primers can be large polynucleotides, such as from about 200 nucleotides to several kilobases or more. Primers may be selected to be “substantially complementary” to the sequence on the template to which it is designed to hybridise and serve as a site for the initiation of synthesis. By “substantially complementary”, it is meant that the primer is sufficiently complementary to hybridise with a target nucleotide sequence. Preferably, the primer contains no mismatches with the template to which it is designed to hybridise but this is not essential. For example, non-complementary nucleotides may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the template.
  • non-complementary nucleotides or a stretch of non-complementary nucleotides can be interspersed into a primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridise therewith and thereby form a template for synthesis of the extension product of the primer.
  • the term "amplification” refers to a biochemical reaction using a ligase or similar enzyme that covalently links two oligonucleotides or two oligonucleotide sub-sequences, such as a ligase chain reaction (LCR).
  • Ligase enzymes ligate the two oligonucleotides or oligonucleotide sub-sequences when they hybridize at adjacent sites in the target nucleic acid sequence.
  • the two oligonucleotides or oligonucleotide subsequences hybridize at sites that are one or more nucleic acid residues apart, i.e., they are not adjacent, then the single stranded region between the double stranded regions is converted to a double stranded region using a polymerase, and the ligase enzyme then links the adjacent oligonucleotides to form a continuous double stranded region.
  • Another embodiment of the diagnostic method of the present invention comprises detecting the presence and/or amount of one or more proteins which are associated with chlamydial infection in a subject, wherein the presence and/or amount of said proteins indicates the presence or stage of chlamydial infection in a subject.
  • protein may be used interchangeably with the terms “peptide” and “polypeptide” herein and refers to a polymer of amino acid residues and to variants of same.
  • protein variant refers to proteins whose sequence is distinguished from a reference protein sequence by substitution, deletion or addition of at least one amino acid.
  • the present invention particularly provides an isolated protein selected from the listing consisting of CT147, CT314, CT727, CT396, CT423, CT157 and CT413.
  • variants may be deduced from other species belonging to the family Chlamydiaceae by isolation of nucleic acid variants by standard protocols known in the art.
  • variants will be at least 50%, preferably at least 55%, more preferably at least 60%, even more preferably at least 65%, even more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90% and still even more preferably at least 95% homologous to a protein of the present invention, for example, CT147 (SEQ ID NO:2), CT314 (SEQ ID NO:4), CT727 (SEQ ID NO:6), CT396 (SEQ ID NO:8), CT423 (SEQ ID NO:10), CT157 (SEQ ID NO:12) and CT413 (SEQ ID NO:13).
  • Reference to "at least 50%” includes at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.
  • CT proteins When used to detect anti-chlamydial antibodies, one or two or three or four or five or six or all seven of the CT proteins may be employed.
  • the present invention includes an isolated antibody (polyclonal, monoclonal or humanized or deimmunized antibody or a fragment thereof to any or all of CT147, CT314, CT727, CT396, CT423, CT157 and/or CT143.
  • Antigen-binding molecules that are specific a protein of the present invention can be used detecting the presence and/or amount of one or more proteins which are associated with chlamydial infection in a subject.
  • antigen-binding molecule refers to a molecule that is specific for, and can therefore form a complex with, a protein, such as an antigen.
  • the term "antigen” is used herein in its broadest sense to refer to a molecule that is capable of reacting in and/or inducing an immune response. Reference to an "antigen” includes an antigenic determinant or epitope.
  • An antigen-binding molecule may be an immunoglobulin molecule.
  • immunoglobulin is used herein to refer to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes.
  • the recognized immunoglobulin genes include the K, ⁇ , ⁇ , ⁇ (IgGi, IgG 2 , IgG 3 , IgG 4 ), ⁇ , ⁇ and ⁇ constant region genes, as well as the myriad of other immunoglobulin variable region genes.
  • One form of immunoglobulin constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions (V L and V H respectively) are together responsible for binding to an antigen, and the constant regions are responsible for the antibody effector functions.
  • immunoglobulins may exist in a variety of other forms including, for example, Fv, scFv, Fab, Fab' and (Fab') 2 forms.
  • the preferred antigen-binding molecules of the present invention are antibodies.
  • the antigen-binding molecules may be polyclonal antibodies.
  • Such antibodies may be prepared, for example, by injecting a protein (e.g., CT147, CT314, CT727, CT396, CT423, CTl 57 and CT413 or fragments thereof) into a production species, which may include mice or rabbits, to obtain polyclonal antisera.
  • a protein e.g., CT147, CT314, CT727, CT396, CT423, CTl 57 and CT413 or fragments thereof.
  • monoclonal antibodies may be produced using the standard method as described, for example, by Kohler and Milstein (Nature 256:495-497, 1975), or by more recent modifications thereof as described, for example, in Coligan et al, "Current Protocols In Immunology” , John Wiley & Sons Inc, 1991, by immortalising spleen or other antibody-producing cells derived from a production species which has been inoculated with the proteins of the present invention.
  • the invention also contemplates as antigen-binding molecules Fv, Fab, Fab'and (Fab') 2 immunoglobulin fragments.
  • the antigen-binding molecule may comprise a synthetic stabilised Fv fragment.
  • Exemplary fragments of this type include single chain Fv fragments (sFv, frequently termed scFv) in which a peptide linker is used to bridge the N terminus or C terminus of a VH domain with the C terminus or N-terminus, respectively, of a VL domain.
  • sFv single chain Fv fragments
  • scFv single chain Fv fragments
  • ScFv lack all constant parts of whole antibodies and are not able to activate complement.
  • Suitable peptide linkers for joining the VH and VL domains are those which allow the VH and VL domains to fold into a single polypeptide chain having an antigen binding site with a three dimensional structure similar to that of the antigen binding site of a whole antibody from which the Fv fragment is derived.
  • Linkers having the desired properties may be obtained by the method disclosed in U.S. Patent No. 4,946,778. However, in some cases a linker is absent.
  • ScFvs may be prepared, for example, in accordance with methods outlined in Kreber et al, (J Immunol. Methods 201:35-55, 1997). Alternatively, they may be prepared by methods described in U.S. Patent No. 5,091,513, European Patent No 239,400 or the articles by Winter and Milstein (Nature 349:293, 1991) and Pl ⁇ nckthun et al, (In "Antibody engineering : A practical approach", 203-252: 1996).
  • the synthetic stabilised Fv fragment comprises a disulphide stabilised Fv (dsFv) in which cysteine residues are introduced into the V H and VL domains such that in the fully folded Fv molecule the two residues will form a disulphide bond therebetween.
  • dsFv disulphide stabilised Fv
  • Suitable methods of producing dsFv are described for example in (Reiter et al, J. Biol. Chem. 262:18327-18331, 1994; Reiter et al, Biochem. 35:5451-5459, 1994; Reiter et al, Cancer Res. 54:2714-2718, 1994 and Webber et al, MoI. Immunol. 32:249-258, 1995).
  • antigen-binding molecules are single variable region domains (termed dAbs) as for example disclosed in (Ward et al, Nature 341:544-546, 1989; Hamers-Casterman et al, Nature 3(53:446-448, 1993 and Davies & Riechmann, FEBS Lett. 339:285-290, 1994).
  • the antigen-binding molecule may comprise a "minibody".
  • minibodies are small versions of whole antibodies, which encode in a single chain the essential elements of a whole antibody.
  • the minibody is comprised of the V H and V L domains of a native antibody fused to the hinge region and CH3 domain of the immunoglobulin molecule as, for example, disclosed in U.S. Patent No 5,837,821.
  • the antigen binding molecule may comprise non- immunoglobulin derived, protein frameworks.
  • non- immunoglobulin derived, protein frameworks For example, reference may be made to Ku & Schultz (Proc Natl Acad Sci USA 92:652-6556, 1995) which discloses a four-helix bundle protein cytochrome b562 having two loops randomised to create complementarity determining regions (CDRs), which have been selected for antigen binding.
  • the antigen-binding molecule may be multivalent (i.e. having more than one antigen- binding site). Such multivalent molecules may be specific for one or more antigens. Multivalent molecules of this type may be prepared by dimerisation of two antibody fragments through a cysteinyl-containing peptide as, for example disclosed by (Adams et al, Cancer Res. 55:4026-4034, 1993; Cumber et al, J. Immunol. 149:120-126, 1992). Alternatively, dimerisation may be facilitated by fusion of the antibody fragments to amphiphilic helices that naturally dimerise (Plunckthun, Biochem.
  • the multivalent molecule may comprise a multivalent single chain antibody (multi-scFv) comprising at least two scFvs linked together by a peptide linker.
  • multi-scFv multivalent single chain antibody
  • non-covalently or covalently linked scFv dimers termed "diabodies" may be used.
  • Multi-scFvs may be bispecific or greater depending on the number of scFvs employed having different antigen binding specificities. Multi-scFvs may be prepared for example by methods disclosed in U.S. Patent No. 5,892,020.
  • the above antigen-binding molecules have utility in detecting directly or indirectly the presence and/or amount of one or more proteins which are associated with chlamydial infection in a subject, such as CT147, CT314, CT727, CT396, CT423, CT157 and CT413, through techniques such as enzyme-linked immunosorbent assays (ELISAs) and Western blotting.
  • Illustrative assay strategies which can be used to detect a protein of the invention include, but are not limited to, immunoassays involving the binding of an antigen-binding molecule to the protein (e.g., CT147, CT314, CT727, CT396, CT423, CT157 and CT413) in the sample, and the detection of a complex comprising the antigen-binding molecule and the protein.
  • Preferred immunoassays are those that can measure the presence and/or amount of a protein according to the present invention.
  • an antigen-binding molecule that is specific for a protein of the invention is contacted with a biological sample suspected of containing said protein.
  • the biological sample is suitably a specimen, which is suspected of containing a chlamydial organism or antibodies.
  • biological samples include serum, whole blood, urine and secretions or washings.
  • the biological sample may comprise fallopian tube washings from infertile women.
  • concentration of a complex comprising the antigen-binding molecule and the target polypeptide is measured and the measured complex concentration is then related to the concentration of target polypeptide in the sample.
  • concentration of said polypeptide is compared to a reference or baseline level of said polypeptide corresponding to the lytic phase of the developmental cycle of a chlamydial species under test. The presence of the persistent phase is detected or a chronic chlamydial infection is diagnosed if the concentration of the polypeptide corresponds to a non-reference level concentration.
  • an antigen-binding molecule according to the invention having a reporter molecule associated therewith may be utilised in immunoassays.
  • immunoassays include, but are not limited to, radioimmunoassays (RIAs), ELISAs and immunochromatographic techniques (ICTs), Western blotting which are well known those of skill in the art.
  • RIAs radioimmunoassays
  • ICTs immunochromatographic techniques
  • Western blotting which are well known those of skill in the art.
  • Coligan et al "Current Protocols In Immunology", John Wiley & Sons Inc, 1991 which discloses a variety of immunoassays that may be used in accordance with the present invention.
  • Immunoassays may include competitive assays as understood in the art or as for example described infra. It will be understood that the present invention encompasses qualitative and quantitative immunoassays. Suitable immunoassay techniques are described for example in U.S. Patent Nos. 4,016,043; 4,424, 279 and 4,018,653. These include both single-site and two-site assays of the non-competitive types, as well as the traditional competitive binding assays. These assays also include direct binding of a labeled antigen- binding molecule to a target antigen.
  • an unlabeled antigen-binding molecule such as an unlabeled antibody is immobilised on a solid substrate and the sample to be tested brought into contact with the bound molecule.
  • Another antigen-binding molecule suitably a second antibody specific to the antigen, labeled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labeled antibody. Any unreacted material is washed away and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may be either qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of antigen.
  • the forward assay includes a simultaneous assay, in which both sample and labeled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including minor variations as will be readily apparent.
  • the sample is one that might contain an antigen including a tissue or fluid as described above.
  • An alternative method involves immobilising the antigen in the biological sample and then exposing the immobilised antigen to specific antibody that may or may not be labeled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound antigen may be detectable by direct labelling with the antibody. Alternatively, a second labeled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.
  • the reporter molecule associated with the antigen-binding molecule may include the following: (a) direct attachment of the reporter molecule to the antigen-binding molecule; (b) indirect attachment of the reporter molecule to the antigen-binding molecule; i.e., attachment of the reporter molecule to another assay reagent which subsequently binds to the antigen-binding molecule; and (c) attachment to a subsequent reaction product of the antigen-binding molecule.
  • the reporter molecule may be selected from a group including a chromogen, a catalyst, an enzyme, a fluorochrome, a chemiluminescent molecule, a lanthanide ion such as Europium (Eu34), a radioisotope and a direct visual label.
  • a colloidal metallic or non- metallic particle a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like.
  • Suitable enzymes suitable for use as reporter molecules is disclosed in U.S. Patent Nos. 4,366,241; 4,843,000 and 4,849,338.
  • Suitable enzymes useful in the present invention include alkaline phosphatase, horseradish peroxidase, luciferase, p-galactosidase, glucose oxidase, lysozyme, malate dehydrogenase and the like.
  • the enzymes may be used alone or in combination with a second enzyme that is in solution.
  • Suitable fluorocliromes include, but are not limited to, fluorescein isothiocyanate (PITC), tetramethylrhodamine isothiocyanate (TRITC), R-Phycoerythrin (RPE), and Texas Red.
  • PITC fluorescein isothiocyanate
  • TRITC tetramethylrhodamine isothiocyanate
  • RPE R-Phycoerythrin
  • Texas Red Texas Red
  • Other exemplary fluorochromes include those discussed by International Patent Publication No. WO 93/06121. Reference also may be made to the fluorochromes described in U.S. Patent Nos. 5,573,909 and 5,326,692. Alternatively, reference may be made to the fluorochromes described in U.S. Patent Nos.
  • fluorogenic substrates which yield a fluorescent product rather than the chromogenic substrates noted above.
  • the enzyme-labeled antibody is added to the first antibody-antigen complex. It is then allowed to bind, and excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of antigen which was present in the sample.
  • fluorescent compounds such as fluorescein, rhodamine and the lanthanide, europium (EU) may be chemically coupled to antibodies without altering their binding capacity.
  • the fluorochrome-labeled antibody When activated by illumination with light of a particular wavelength, the fluorochrome-labeled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope.
  • the fluorescent-labeled antibody is allowed to bind to the first antibody-antigen complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to light of an appropriate wavelength. The fluorescence observed indicates the presence of the antigen of interest.
  • Immunofluorometric assays IFMA
  • other reporter molecules such as radioisotope, chemiluminescent or bioluminescent molecules may also be employed.
  • CT314, CT727, CT396, CT423, CT157 and CT413) levels are available, including, for instance, those involving testing for an altered level of the target polypeptide binding activity to the target polypeptide binding partner, or Western blot analysis of target protein levels in tissues, cells or fluids using anti-target protein antigen-binding molecules, or assaying the amount of antigen-binding molecule or other target polypeptide binding partner which is not bound to a sample, and subtracting from the total amount of antigen- binding molecule or binding partner added.
  • the presence of a chlamydial infection in a subject may also be determined by assaying a subjects immune response to chlamydial antigens. Therefore, another embodiment of the diagnostic method of the present invention comprises detecting the presence and/or amount of one or more antigen-binding molecules specific for one or more proteins which are associated with chlamydial infection in a subject, wherein the presence and/or amount of said antigen-binding molecules indicates the presence or stage of chlamydial infection in a subject.
  • the preferred antigen-binding molecules of the present invention are antibodies, and in accordance with this embodiment, the antibodies are obtained from a biological sample of a subject.
  • the biological sample is blood, plasma or serum.
  • the antigen-molecules of the present invention are specific for one or more proteins which are associated with chlamydial infection in a subject. Such proteins are preferably chlamydial antigens.
  • the present invention provides a method for determining the presence or stage of chlamydial infection in a subject, said method comprising:
  • chlamydial antigens refers to one or more antigens that are associated with chlamydial infection in a subject. Such antigens elicit an immune response in the subject, thereby generating in said subject the antigen-binding molecules which are predictive of the presence or stage of a chlamydial infection.
  • the chlamydial antigens are derived from the organism that causes the chlamydial infection.
  • the chlamydial antigens are selected from the group comprising CT147, CT314, CT727, CT396, CT423, CTl 57 and CT413.
  • the present invention contemplates a method for determining the presence or stage of chlamydial infection in a subject, said method comprising detecting the presence and/or amount of antibodies in a biological sample from said subject to one or more proteins selected from the list consiting of CT 147 (SEQ ID NO:2), CT314 (SEQ ID NO:4), CT727 (SEQ ID NO:6), CT396 (SEQ ID NO:8), CT423 (SEQ ID NO: 10), CT157 (SEQ ID NO:12) and CT413 (SEQ ID NO:13) wherein the presence of antibodies to one or more of said protein indicates the presence or stage of chlamydial infection in said subject.
  • All the essential components required for determining the presence or stage of chlamydial infection in a subject according to the methods of the present invention may be assembled together in a kit.
  • the present invention also provides, therefore, a kit suitable for use with a method for determining the presence or stage of chlamydial infection in a subject, said kit comprising components which facilitate the detection of the presence and/or amount of one or more proteins or variants thereof which are associated with chlamydial infection in a subject, or one or more nucleic acids or variants thereof encoding said proteins, one or more antigen- binding molecules specific for said proteins.
  • the kit of the present invention comprises all the required nucleic acid primers, nucleotide precursors, enzymes, buffer solutions, antigen-binding molecules and the like.
  • the kit of the present invention comprises one or more chlamydial antigens.
  • the chlamydial antigens are provided in a recombinant form.
  • One or two or three or four or five or six or seven of CT 147, CT314, CT727, CT396, CT423, CT157 and or CT413 may be present in the kit.
  • kits for identifying a chlamydial infection or for distinguishing between strains of Chlamydia comprising a support or container adapted to contain one or more proteins selected from CT 147, CT314, CT727, CT396, CT423, CT157 or CT413, said proteins, said support or contain capable of receiving a biological sample potentially comprising antibodies to one or more of said proteins.
  • a recombinant chlamydial antigen, or fragment thereof may be prepared by any suitable procedure known to those of skill in the art.
  • a recombinant chlamydial antigen may be prepared by a procedure including the steps of (a) preparing a recombinant nucleic acid comprising a nucleotide sequence encoding a protein comprising the sequence of, for example, CT147 (SEQ ID NO:2), CT314 (SEQ ID NO:4), CT727 (SEQ ID NO:6), CT396 (SEQ ID NO:8), CT423 (SEQ ID NO:10), CT157 (SEQ ID NO:12) and CT413 (SEQ ID NO: 13), or a biologically active fragment thereof, or variant of these, which nucleotide sequence is operably linked to regulatory elements; (b) introducing the recombinant nucleic acid into a suitable host cell; (c) culturing the host cell to express recombinant antigen from said recombin
  • nucleic acid sequences of the present invention may be determined by conventional techniques.
  • a nucleic acid sequence according to any one of, for example, CT147 (SEQ ID NO:1), CT314 (SEQ ID NO:3), CT727 (SEQ ID NO:5), CT396 (SEQ ID NO:7), CT423 (SEQ ID NO:9), CTl 57 (SEQ ID NO:11) and CT413 (SEQ ID NO:13) can be mutated using random mutagenesis (e.g., transposon mutagenesis), oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis and cassette mutagenesis as is known in the art.
  • suitable nucleic acid sequence variants of the invention may be prepared according to the following procedure: creating primers which are optionally degenerate wherein each comprises a portion of a reference nucleic acid sequence encoding a reference protein or fragment of the invention, for example CT147 (SEQ ID NO:2), CT314 (SEQ ID NO:4), CT727 (SEQ ID NO:6), CT396 (SEQ ID NO:8), CT423 (SEQ ID NO: 10), CT157 (SEQ ID NO:12) and CT413 (SEQ ID NO:13); obtaining a nucleic acid extract from an organism, which is preferably an animal, and more preferably a mammal; and using said primers to amplify, via nucleic acid amplification techniques, at least one amplification product from said nucleic acid extract, wherein said amplification product corresponds to a nucleic acid variant.
  • primers which are optionally degenerate wherein each comprises a portion of a reference nucleic acid sequence encoding
  • the recombinant nucleic acid sequence is preferably in the form of an expression vector that may be a self-replicating extra-chromosomal vector such as a plasmid, or a vector that integrates into a host genome.
  • vector is meant a nucleic acid molecule, preferably a
  • DNA molecule derived, for example, from a plasmid, bacteriophage, or plant virus, into which a nucleic acid sequence may be inserted or cloned.
  • a vector preferably contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible.
  • the vector may be an autonomously replicating vector i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.
  • the vector may contain any means for assuring self-replication.
  • the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome (s) into which it has been integrated.
  • a vector system may comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the vector may also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants. Examples of such resistance genes are well known to those of skill in the art
  • the regulatory elements will generally be appropriate for the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells. Typically, the regulatory elements include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are contemplated by the invention. The promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter.
  • the expression vector contains a selectable marker gene to allow the selection of transformed host cells.
  • Selection genes are well known in the art and will vary with the host cell used.
  • the expression vector may also include a fusion partner (typically provided by the expression vector) so that the recombinant polypeptide of the invention is expressed as a fusion polypeptide with said fusion partner.
  • a fusion partner typically provided by the expression vector
  • the main advantage of fusion partners is that they assist identification and/or purification of said fusion polypeptide.
  • fusion partners include, but are not limited to, glutathione-S-transferase (GST), Fc potion of human IgG, maltose binding protein (MBP) and hexahistidine (HIS6), which are particularly useful for isolation of the fusion polypeptide by affinity chromatography.
  • GST glutathione-S-transferase
  • MBP maltose binding protein
  • HIS6 hexahistidine
  • relevant matrices for affinity chromatography are glutathione-, amylose-, and nickel-or cobalt-conjugated resins respectively.
  • Many such matrices are available in "kit” form, such as the QlAexpress system (Qiagen) useful with (HIS6) fusion partners and the Pharmacia GST purification system.
  • the recombinant polynucleotide is expressed in the commercial vector pFLAG as described more fully hereinafter.
  • Another fusion partner well known in the art is green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • This fusion partner serves as a fluorescent "tag" which allows the fusion polypeptide of the invention to be identified by fluorescence microscopy or by flow cytometry.
  • the GFP tag is useful when assessing subcellular localisation of the fusion polypeptide of the invention, or for isolating cells which express the fusion polypeptide of the invention.
  • Flow cytometric methods such as fluorescence activated cell sorting (FACS) are particularly useful in this latter application.
  • the fusion partners also have protease cleavage sites, such as for Factor Xa or Thrombin, which allow the relevant protease to partially digest the fusion polypeptide of the invention and thereby liberate the recombinant polypeptide of the invention therefrom.
  • the liberated polypeptide can then be isolated from the fusion partner by subsequent chromatographic separation.
  • Fusion partners according to the invention also include within their scope "epitope tags", which are usually short peptide sequences for which a specific antibody is available.
  • epitope tags for which specific monoclonal antibodies are readily available include c-Myc, influenza virus, haemagglutinin and FLAG tags.
  • the vector is pPROEx (Life Technologies).
  • the step of introducing into the host cell the recombinant nucleic acid sequence may be effected by any suitable method including transfection, and transformation, the choice of which will be dependent on the host cell employed. Such methods are well known to those of skill in the art.
  • Recombinant proteins of the present invention may be produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding a polypeptide, biologically active fragment, variant or derivative according to the invention.
  • the conditions appropriate for protein expression will vary with the choice of expression vector and the host cell. This is easily ascertained by one skilled in the art through routine experimentation.
  • Suitable host cells for expression may be prokaryotic or eukaryotic.
  • One preferred host cell for expression of a polypeptide according to the invention is a bacterium.
  • the bacterium used may be Escherichia coli.
  • the host cell may be an insect cell such as, for example, SF9 cells that may be utilised with a baculovirus expression system.
  • the recombinant protein may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook et al, "Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Press, 1989; Ausubel et al, "Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998.
  • the polypeptide, fragment, variant or derivative may be synthesised using solution synthesis or solid phase synthesis as described, for example, in Roberge et al, ⁇ Science 269:202-204, 1995).
  • the chlamydial antigens in the form of recombinant protein, may be provided immobilised on a solid substrate. Suitable substrates and immobilisation methods would be known to a person skilled in the art.
  • the solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the solid supports may be in the form of tubes, beads, discs or microplates, or any other surface suitable for conducting an immunoassay.
  • the binding processes are well known in the art and generally consist of cross-linking, covalently binding or physically adsorbing, the polymer-antibody complex to the solid support, Alternatively, the chlamydial antigens may be provided free in solution.
  • the present invention also provides a method for identifying proteins, nucleic acids and antigen-binding molecules associated with chlamydial infection in a subject which are suitable for use with the diagnostic methods and kits described herein.
  • the present invention is directed to the use of a protein selected from CTl 47, CT314, CT727, CT396, CT423, CT157 and CT413 in the manufacture of a diagnostic agent for chlamydial infection.
  • proteins or nucleic acid sequences identified in accordance with the present invention are also useful in the development of methods and agents for preventing and/or treating chlamydial infection in a subject, such as, but not limited to, immunotherapeutic methods and agents.
  • the present invention provides, therefore, a method for preventing and/or treating chlamydial infection in a subject said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient to prevent and/or treat chlamydial infection in said subject.
  • the present invention further provides a vaccine against chalymidal infection said vaccine comprising at least one protein selected from CT147, CT314, CT727, CT396, CT423, CTl 53 and CT413 or an antigenic fragment thereof said vaccine further comprising one or more pharmaceutically acceptable carriers, diluents, excipients, adjuvants and/or immune response enhancers.
  • the present invention contemplates a method for vaccinating a subject against chlamydial infection said method comprising administering to said subject an antibody- inducing effective amount of one or more proteins selected from CT147, CT314, CT727, CT396, CT423, CT157 and CT413 or an immunogenic fragment thereof.
  • treatment may mean a reduction in the severity of an existing condition.
  • treatment is also taken to encompass “prophylactic treatment” to prevent the onset of a condition.
  • treatment does not necessarily imply that a subject is treated until total recovery.
  • prophylactic treatment does not necessarily mean that the subject will not eventually contract a condition.
  • the agent is an immunotherapeutic agent that is in the form of an immunotherapeutic composition such as, but not limited to, a vaccine.
  • Suitable agents include one or more of CT314, CT147, CT727, CT396, CT157, CT423 and/or CT413 or antigenic homologs or fragments thereof.
  • Such a composition may be prepared using routine methods known to persons skilled in the art. Exemplary procedures include, for example, those described in Levine et al, "New Generation Vaccines", Marcel Dekker Inc, 1997.
  • immunotherapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified.
  • the active immunogenic ingredients are often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient.
  • excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the immunotherapeutic composition or vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants that enhance the effectiveness of the composition.
  • a polypeptide, fragment, variant or derivative of the invention according to the invention can be mixed, conjugated or fused with other antigens, including B or T cell epitopes of other antigens. In addition, it can be conjugated to a carrier as described below.
  • a haptenic peptide When a haptenic peptide is used (i.e., a peptide which reacts with cognate antibodies, but cannot itself elicit an immune response), it can be conjugated with an immunogenic carrier.
  • immunogenic carriers include, for example, thyroglobulin; albumins such as human serum albumin; toxins, toxoids or any mutant crossreactive material (CRM) of the toxin from tetanus, diphtheria, pertussis, Pseudomonas, E.
  • coli Staphylococcus and Streptococcus
  • polyamino acids such as poly(lysine: glutamic acid); influenza; Rotavirus VP6, Parvovirus VPl and VP2; hepatitis B virus core protein; hepatitis B virus recombinant vaccine and the like.
  • a fragment or epitope of a carrier protein or other immunogenic protein may be used.
  • a haptenic peptide can be coupled to a T cell epitope of a bacterial toxin, toxoid or CRM.
  • U.S. Patent No 5,785,973 U.S. Patent No 5,785,973.
  • a polypeptide, fragment, variant or derivative of the invention may act as a carrier protein in vaccine compositions directed against an organism of the Chlamydiaceae family.
  • the immunotherapeutic compositions of the invention may be administered as multivalent subunit compositions or vaccines in combination with other chlamydial immunogens such as MOMP. Alternatively, or additionally, they may be administered in concert with immunologically active antigens against other pathogenic species such as, for example, the pathogenic bacteria H. influenzae, M. catarrhalis, N. gonorrhoeae, E. coli, S. pneumoniae etc.
  • the immunotherapeutic compositions may include an adjuvant as is well known in the art.
  • Suitable adjuvants include, but are not limited to: surface active substances such as hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyldioctadecylammonium bromide, N, N-dicoctadecyl-N', N'bis (2-hydroxyethyl- propanediamine), methoxyhexadecylglycerol, and pluronic polyols; polyamines such as pyran, dextransulfate, poly IC carbopol; peptides such as muramyl dipeptide and derivatives such as N-acetyl-muramyl-L-threonyl-D-isoglutamine (thur-MDP), N-acetyl- nor-muramyl-L-alanyl-
  • lymphokines and QuilA.
  • the effectiveness of an adjuvant may be determined for example by measuring the amount of antibodies resulting from the administration of the composition, wherein those antibodies are directed against one or more said chlamydial antigens or by measuring antigen specific T cell proliferation or cytolytic activity.
  • the immunotherapeutic composition may be administered via a mucosal route such as, but not limited to, orally, urogenitally or transdermally or combination of these.
  • the adjuvant is preferably a mucosal adjuvant.
  • the mucosal adjuvant is cholera toxin or diphtheria toxin.
  • Mucosal adjuvants other than cholera toxin or diphtheria toxin which may be used in accordance with the present invention include non-toxic derivatives of said toxins, such as the B sub-unit (CTB), chemically modified cholera or diphtheria toxin, or related proteins produced by modification of the cholera toxin or diphtheria toxin amino acid sequence. These may be added to, or conjugated with, the polypeptides, fragments, variants or derivatives of the invention.
  • CTB B sub-unit
  • the same techniques can be applied to other molecules with mucosal adjuvant or delivery properties such as E. coli heat labile toxin.
  • mucosal adjuvant or delivery activity may be used such as bile; polycations such as DEAE-dextran and polyornithine ; detergents such as sodium dodecyl benzene sulphate; lipid-conjugated materials ; antibiotics such as streptomycin; vitamin A; and other compounds that alter the structural or functional integrity of mucosal surfaces.
  • Other mucosally active compounds include derivatives of microbial structures such as MDP; acridine and cimetidine.
  • the immunogenic agents of the invention may be delivered in ISCOMS (immune stimulating complexes), ISCOMS containing CTB, liposomes or encapsulated in compounds such as acrylates or poly (DL-lactide-co-glycoside) to form microspheres of a size suited to adsorption by M cells.
  • ISCOMS immune stimulating complexes
  • ISCOMS containing CTB liposomes or encapsulated in compounds such as acrylates or poly (DL-lactide-co-glycoside) to form microspheres of a size suited to adsorption by M cells.
  • micro or nanoparticles may be covalently attached to molecules, which have specific epithelial receptors.
  • the polypeptide, fragments, variant or derivative of the invention may also be incorporated into oily emulsions and delivered orally.
  • An extensive though not exhaustive list of adjuvants can be found in Cox and Coulter, "Advances in adjuvant technology and application", In “Anim
  • polypeptides, fragments, variants or derivatives of the invention may also be expressed by attenuated viral hosts.
  • a virus may be rendered substantially avirulent by any suitable physical (e.g., heat treatment) or chemical means (e.g., formaldehyde treatment).
  • Attenuated viral hosts may comprise live viruses or inactivated viruses.
  • Attenuated viral or bacterial hosts which may be useful in a vaccine according to the invention may comprise viral vectors inclusive of adenovirus, cytomegalovirus and preferably pox viruses such as vaccinia (see, for U.S. Patent No. 4,603,112) and attenuated Salmonella strains (see, for example U.S. Patent No. 4,550,081).
  • viral vectors inclusive of adenovirus, cytomegalovirus and preferably pox viruses such as vaccinia (see, for U.S. Patent No. 4,603,112) and attenuated Salmonella strains (see, for example U.S. Patent No. 4,550,081).
  • Live vaccines are particularly advantageous because they lead to a prolonged stimulus that can confer substantially long-lasting immunity.
  • these agents may be delivered to the host using a live vaccine vector, in particular using live recombinant bacteria, viruses or other live agents, containing the genetic material necessary for the expression of the polypeptide, fragment, variant or derivative of the invention as a foreign antigen.
  • Multivalent immunotherapeutic compositions or vaccines can be prepared from one or more organisms of the Chlamydiaceae family that express different phase antigens or epitopes.
  • epitopes of other pathogenic microorganisms can be incorporated into the compositions.
  • this will involve the construction of a recombinant vaccinia virus to express a nucleic acid sequence according to the invention.
  • the recombinant vaccinia virus Upon introduction into a host, the recombinant vaccinia virus expresses the immunogenic agent, and thereby elicits a host CTL response.
  • U.S. Patent No. 4,722,848 describes vaccinia vectors and methods useful in immunisation protocols.
  • a variety of other vectors useful for therapeutic administration or immunisation with the immunogenic agents of the invention will be apparent to those skilled in the art from the present disclosure.
  • a polynucleotide of the invention may be used as a vaccine in the form of a "naked DNA" vaccine as is known in the art.
  • an expression vector of the invention may be introduced into a mammal, where it causes production of a polypeptide in vivo, against which the host mounts an immune response as for example described in Barry et al, ⁇ Nature 377:632-635, 1995).
  • nucleic acid-based immunotherapeutic compositions comprising an expression vector including a polynucleotide encoding an at least one chlymidial antigen, wherein said polynucleotide is operably linked to a regulatory polynucleotide, together with a pharmaceutically acceptable carrier.
  • nucleic acid based compositions all modes of delivery of such compositions are contemplated by the present invention. Delivery of these compositions to cells or tissues of an animal may be facilitated by microprojectile bombardment, liposome mediated transfection (e.g., lipofectin or lipofectamine), electroporation, calcium phosphate or DEAE-dextran-mediated transfection, for example.
  • a synthetic construct may be used as a therapeutic or prophylactic composition in the form of a "naked DNA" composition as is known in the art.
  • suitable delivery methods may be found in Ausubel et al, "Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998.
  • the compositions may be administered by intradermal (e.g., using panjet (trademark) delivery) or intramuscular routes.
  • the immunotherapeutic compositions will suitably elicit a B cell response and preferably a T cell response.
  • Immunotherapeutic compositions which produce a desired immune response can be evaluated using animal models of chlamydial infection (e.g., mouse for both urogenital and respiratory and cardiovascular infections; guinea pig for predominantly urogenital infections).
  • the selected animal model is suitably be vaccinated (e.g., via several mucosal routes) using either full length recombinant proteins or portions thereof and boosted after 4-6 weeks.
  • the immune response (preferably both antibody and cell mediated) is typically measured at weekly intervals. Generally, after periods of 8 weeks and 6 months, the vaccinated as well as unvaccinated control animals, are challenged with live Chlamydia.
  • the immune responses (preferably both antibody and cell mediated) are continued to be measured at weekly intervals. Typically, several animals from each group are sacrificed and the status of disease evaluated, after 3 and 6 months and compared with unvaccinated controls.
  • the agents and/or immunotherapeutic compositions defined in accordance with the present invention may be co-administered with one or more other agents and/or immunotherapeutic compositions.
  • Reference herein to "co-administered” means simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes.
  • Reference herein to "sequential" administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two types of agents and/or immunotherapeutic compositions. Co-administration of the agents and/or immunotherapeutic compositions may occur in any order.
  • the methods, kits and agents contemplated by the present invention may be used in relation to any infection caused by organisms of the Chlamydiaceae family.
  • the Chlamydiaceae family comprises two distinct genera, Chlamydia and Chlamydophila.
  • the Chlamydia genus comprises the species C. muridarum, C. suis, C. trachomatis.
  • the Chlamydophila genus comprises the species C. abortus, C. caviae, C. felia, C. pecorum, C. pneumoniae, and C. psittaci.
  • the infection is caused by C. trachomatis.
  • the present invention also enables discrimination between C. pneumoniae and C. trachomitis or between strains of either such as C. trachomitis strain, C, D, K or L2.
  • the present invention is further directed to the use of a protein selected from CTl 47, CT314, CT727, CT396, CT423, CT157 and CT413 in the manufacture of a medicament to treat chlamydial infection.
  • kits and agents contemplated by the present invention are also useful in relation to conditions that are related to, or otherwise arise from, chlamydial infection such as a disease of the systemic vasculature (e.g. heart and lung disease).
  • chlamydial infection such as a disease of the systemic vasculature (e.g. heart and lung disease).
  • Patient Groups Samples of the following clinical populations were obtained from women attending the Brisbane sexual Health Clinic for first-time or follow-up diagnosis and management of C. trachomatis infections diagnosed by urine or swab based Amplicor PCR (Roche Diagnostics).
  • Controls (women with endometriosis not due to C. trachomatis infection). Samples were obtained from women presenting to the Wesley Hospital Department of Reproductive Medicine (Brisbane, Australia) for laparoscopic and falloposcopic investigation of infertility with diagnosis based on surgical findings. Infertile Control patients visually diagnosed with endometriosis, with no previous history or positive C trachomatis serology (n - 18).
  • the "204" group referred to in the examples is a group of subjects with a second acute Chlamydia infection.
  • the "212" group is a group of subjects twelve months subsequent to reinfection.
  • C. trachomatis Cell Culture HEp-2 cells were cultured in 5% FCS-DMEM, supplemented with 5% heat-inactivated foetal calf serum (FCS), 0.002% gentamycin, 5% CO 2 and infected with 2mL of C. trachomatis Ul strain. Six hours post infection (pi), media were discarded and 1OmL of fresh 5% v/v FCS-DMEM were added. At 30 hours pi, Chlamydia plus host cells were extracted as follows. Trypsinised cells were resuspended in 48mL phosphate buffered saline (PBS), centrifuged at 1000 rpm for 5 minutes at 4°C and pelleted.
  • PBS phosphate buffered saline
  • the pellet was resuspended in 3mL of PBS, repelleted for 15 seconds, and resuspended in 3mL 2X sample buffer (1% v/v glycerol, 20% w/v SDS, 0.5% v/v beta-mercaptoethanol, IM Tris Cl pH 6.8, bromophenol blue) and stored at -2O 0 C until required for use in SDS- PAGE/western blot experiments.
  • 2X sample buffer 1% v/v glycerol, 20% w/v SDS, 0.5% v/v beta-mercaptoethanol, IM Tris Cl pH 6.8, bromophenol blue
  • chlamydial antigens patient sera from each group were screened against Chlamydia/host cell proteins from non-infected HEp-2 cells (UI) and HEp-2 cells infected with C. trachomatis serovar L2 (I). Briefly, 2 ⁇ L of each protein extract was loaded onto 12.5% w/v polyacrylamide SDS-PAGE gels and electrophoresed at 110 volts for 100 minutes at room temperature (RT).
  • the membrane was incubated with the secondary antibody, conjugated rabbit anti-human horseradish peroxidase (HRP) IgG (Roche) diluted 1:4000 in SM-PBS-T at RT for 1 hour with shaking. Membranes were washed 4X for 15 minutes in PBS-T then detected via chemiluminescence (Amersham Biosciences ECL Plus Detection Kit).
  • HRP horseradish peroxidase
  • the coupled lysate/antibody mix was directly added to 200 ⁇ L buffered protein G suspension and incubated with rotation for 2 hours at 4°C.
  • the mix was pelleted by centrifugation (600Og for 40 seconds), resuspended in 1 mL of wash buffer (5OmM Tris pH 7.6, 15OmM NaCl, 0.1% NP40, 0.03% w/v SDS) and then centrifuged at 1200Og for 30 seconds. The supernatant was discarded and the pellet washed twice in wash buffer before adding 50 ⁇ L of 2X sample buffer giving a final volume of ⁇ 100 ⁇ L. The sample was then heated at 95°C for 10 minutes and stored at - 20°C until required.
  • SDS-PAGE/ Western Blot of Immimoprecipitated Chlamydial Proteins SDS-PAGE/ Western Blot of Immimoprecipitated Chlamydial Proteins:
  • Proteins precipitated by the protein G beads were separated using a 12.5% w/v polyacrylamide SDS-PAGE gel and transferred at 4 0 C, in CAPs buffer, to immobilon-P Polyvinylidene Fluoride (PVDF) membrane prepared as per manufacturer's instructions (Amersham Biosciences).
  • PVDF Polyvinylidene Fluoride
  • a small segment of the membrane containing one marker lane and a section of the imniunoprecipitated proteins was reserved for western blotting whilst the remaining membrane was stained with 0.1% w/v Coomassie (Ig coomassie blue, 40% v/v methanol and 10% glacial acetic acid) for 30 minutes.
  • the coomassie stained membrane was washed in 90% methanol until all background staining was removed.
  • Western blotting was performed on the small membrane segment as per the protocol previously described.
  • the coomassie stained membrane and western blotted section were aligned and compared to identify differential chla
  • N-Terminal Sequencing From the coomassie stained PVDF membrane, potential antigenic bands were excised and forwarded to the Australian Proteome Analysis Facility (APAF) at Macquarie University in Sydney for protein identification. Protein samples attached to PVDF membrane were subjected to 7 cycles of N-terminal sequencing via automated Edman degradation (Applied Biosystems 494 Procise Protein Sequencing System). A lOpmol ⁇ -lactoglobulin standard was used to verify sequencer performance.
  • bands were cut into small pieces and placed into pre-washed (50% v/v acetonitrile (ACN)/0.1% v/v trifluoroacetic acid (TFA)) eppendorf tubes.
  • ACN v/v acetonitrile
  • TSA trifluoroacetic acid
  • Figure 1 shows the typical western blot profiles when sera from acute, recovered acute, acute multiply infected, PID and negative control groups were used to probe uninfected (UI) and infected (I) cell preparations.
  • Four bands, A (>113kDa), B (72.4kDa), C (44.6kDa) and D (13.5kDa) were differentially detected by various patient sera, with the majority of patients responsive to not less than 2 of the 4 antigens (Table 2). Of the four bands, the most commonly reactive were C (71%) and D (74%).
  • Band A was present in 10% of PID patients, compared to 46% of acute, 63% of recovered acute and 67% of acute multiply infected patients.
  • a single negative control patient verified C.
  • Antibody levels to C. pneumoniae and C. trachomatis for all groups were determined by MIF and EIA respectively prior to western blot analysis. Twenty-five percent of acute multiply infected patients demonstrated antibodies to C. pneumoniae. The acute, recovered acute, PID and negative control groups all displayed similar (45-60%) antibody titres. Excluding the negative control group, antibody reactivity to C. trachomatis for the remaining groups ranged between 62% and 100%. Table 2: Patient antibody reactivity and estimated molecular weights of the four identified bands with positive C. trachomatis (C.tr) and C. pneumoniae (C.pn) serology for each patient group.
  • PID (K IO) 10 80 80 100 80 60
  • band A could not be identified via N-terminal sequencing as the protein was not successfully precipitated from the C. trachomatis host-cell lysate.
  • MS analysis of the band A sample identified two possible candidate proteins: CT147 (Conserved Hypothetical Protein - 162.IkDa) and CT314 (DNA-directed RNA polymerase beta chain - 154.9kDa (Table 3).
  • CT147 Consed Hypothetical Protein - 162.IkDa
  • CT314 DNA-directed RNA polymerase beta chain - 154.9kDa (Table 3).
  • CT727 Metal transport P-type ATPase - 70.5kDa
  • TIGR Chlamydia microbial
  • Table 3 Identification of bands A, B and C by N-terminal sequencing or mass spectrometry produced two potential candidates for each bond. Molecular weights and predicted gene function for each are as indicated.
  • trachomatis Ul demonstrated an overall specificity of 63%.
  • Band A showed 47% specificity to C. trachomatis Ul, 27% to C. trachomatis D, 33% to C. trachomatis K and 40% to C. pneumoniae.
  • Band B was well recognised across all chlamydial strains and species tested.
  • Band C demonstrated the highest specificity to C, trachomatis Ul (80%), compared to C. trachomatis D and K strains (both 20%), and C. pneumoniae (47%).
  • Band D revealed 27% reactivity to C. trachomatis K, but 53% and 73% specificity to C. pneumoniae and C. trachomatis Ul respectively.
  • C. trachomatis serovar D did not demonstrate specificity for band D.
  • Panel 4 format (A or B or C or D) displayed the greatest sensitivity of 79% however, the addition of antigen D decreased specificity by 18%.
  • Panel 6 (B + C) demonstrated
  • Acute Patients A orB or C 75 74 58 86
  • Figure 4 shows the typical differential western blot profiles when sera from each male patient group were used to challenge uninfected (UI) and infected (I) cell preparations. Only three of the four bands previously witnessed in female patients were differentially detected by various male sera. Unique to the C. trachomatis infected male patients was an ⁇ 19kDa protein denoted band M. Table 6 shows the percentage prevalence of all four differential bands (B, C, D and M) and the C. trachomatis (EIA) and C. pneumoniae (MIF) serology results for all serum samples. Band A (>113kDa), previously observed in several female patients, was not present in any male group and therefore has been excluded from Table 6.
  • Band B (72.4kDa) was present in 100% of the acute, second acute (204) and chronic groups but only moderately in both the recovered acute and twelve months subsequent to reinfection (212) groups.
  • Band C (44.6kDa) was detected in 36% of the recovered acute and 50% of the 204 patient samples compared to 100% of the remaining C. trachomatis infected patient groups.
  • Two patient groups (212 and chronic) demonstrated no differential profile to band D (13.5kDa), although moderate to high reactivity was observed for the acute, recovered acute and 204 groups. All patient groups, except chronic, showed an high prevalence to the Band M (19kDa; CT413) male marker. Evident in moderate to low levels in the adult male controls were all 4 differential bands.
  • Serological analysis (MIF) revealed a moderate to high incidence of C.
  • Figure 5 shows the coomassie stained gel and accompanying western blot used to specifically target the Band M (19kDa) protein.
  • the arrows indicate the band of interest which was excised for protein identification.
  • N-terminal sequencing returned 7 amino acids (ASAPAAA SEQ ID NO: 14).
  • Table 7 depicts the most significant sequence alignments generated by the NCBI BLASTP program.
  • trachomatis culture preparations used trypsin to detach infected host cells and may have cleaved the PmpB gene into several smaller fragments thereby producing an ⁇ 19kDa protein. Subsequent to an in silico tryptic digest, a 17.3kDa fragment containing the identified seven amino acid sequence was observed (Table 7). Contained within the PmpB fragment is the amino acid motif GGAI, inherent amongst all members of the Pmp family.
  • Species and serovar specificity of the novel male marker was determined by probing C. trachomatis L2, D and K 5 and C. pneumoniae cell extracts with sera from acute, recovered acute, 204, 212 and chronic patients ( Figure 6). Results from the five groups were combined and specificity for bands B, C, D and M were analysed (Table 8). Varied sequence homology is indicated between the C. trachomatis strains and C. pneumoniae as no serovar or species specific band was detected in any of the screened male samples. Band B showed decreased specificity in C. trachomatis serovar D (15%) when compared to C. trachomatis serovars L2 (70%), K (45%) and C. pneumoniae (60%).
  • Figure 7 indicates the reactivities of all four identified bands during various C. trachomatis infection stages.
  • the acute group in the initial stage of infection demonstrates high antibody reactivity to all bands.
  • an overall reduction of reactivity to bands B, C, D and M is observed however, with the advent of a second acute chlamydial infection (204 group), antibody production to all bands is dramatically increased.
  • antigen reactivity for bands D and M has exceeded those of the acute group by 20%. Twelve months subsequent to reinfection (212 group), a 50% decline in antibody levels and the total absence of reactivity to band D is exhibited.
  • Antibody reactivity to band M at this stage of infection is at its lowest level (50%) compared to the other infection phases.
  • Present in the chronic group are antibodies solely to bands B and

Abstract

L'invention concerne, en général, des méthodes de diagnostic et de traitement de maladies infectieuses ou d'autres états associés. Elle concerne, plus particulièrement, des méthodes servant à déterminer la présence d'organismes de la famille Chlamydiaceae chez un sujet, y compris des espèces de Chlamydia, et des méthodes servant à déterminer le degré de l'infection provoquée par ces organismes. Elle concerne également des trousses conçues pour être utilisées avec ces méthodes diagnostiques. Ces méthodes et ces trousses sont particulièrement utiles pour des applications chez l'homme et chez l'animal. Elle concerne, de plus, des méthodes servant à identifier des protéines ou des séquences d'acide nucléique associées à une infection chlamydienne chez un sujet. Ces protéines ou ces séquences d'acide nucléique ne sont pas seulement utiles en ce qui concerne les méthodes diagnostiques décrites par l'invention, mais également pour concevoir des méthodes et des agents servant à prévenir et/ou à traiter l'infection chlamydienne chez un sujet, par exemple, sans limitation, des méthodes et des agents immunothérapeutiques.
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US20100311049A1 (en) * 2005-12-22 2010-12-09 University Of Delhi PCR-Based Kit For Detecting Chlamydia Trachomatis and Nelsseria Gonorrhoeae
US20110256094A1 (en) * 2008-10-09 2011-10-20 Board Of Regents, University Of Texas System Methods and Compositions for Chlamydial Antigens for Diagnosis and Treatment of Chlamydial Infection and Disease

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