WO2007030879A1

WO2007030879A1 - Diagnostic markers and uses therefor

Info

Publication number: WO2007030879A1
Application number: PCT/AU2006/001346
Authority: WO
Inventors: Peter Timms; Trudi Anne Armitage; Thomas Bernard Macnaughton; Terence Patrick Walsh
Original assignee: Diatech Pty Ltd
Priority date: 2005-09-13
Filing date: 2006-09-13
Publication date: 2007-03-22

Abstract

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. Such 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.

Description

DIAGNOSTIC MARKERS AND USES THEREFOR

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

DESCRIPTION OF THE PRIOR ART

Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

Bibliographic details of references provided in this document are listed at the end of the specification. 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).

Current diagnostic tests available for chlamydial infection have wide varying performance characteristics. The most extensively used diagnostic assays are nucleic acid amplification testing (e.g. PCR), antigen detection (direct fluorescent antibody and enzyme immunoassay (EIA)) and serological methods such as microimmunofluorescence (MIF). Diagnosis of Chlamydia-mάucsά infections is routinely performed by PCR as it is sensitive and highly specific. Since testing can only be performed on tissue, urinary or appropriately collected swab specimens and not on patient sera, the use of PCR as a diagnostic tool is somewhat limited. C. trachomatis infections are difficult to diagnose given the asymptomatic nature of infection, thus acute and chronic infection stages cannot be discriminated. Although the serological MIF assay aims to differentiate acute from chronic infections, the decreased specificity as a consequence of cross-reactivity between chlamydial species limits the efficacy of the test (Wong et al, J CHn Path 52:99-102, 1999). Consequently, several new enzyme-linked immunosorbent assays (ELISAs) have incorporated C trachomatis recombinant antigens to improve species specificity (Labsystems Research Laboratory, Helsinki, Finland; Medac, Hamburg, Germany). The Labsystems ELISA employs synthetically derived C. trachomatis-specific epitopes of the major outer membrane protein (MOMP), whilst the Medac ELISA utilises a total Chlamydia-specific lipopolysaccharide (LPS) 3-deoxy-D-mα««σ-2-octulopyranosonic recombinant fragment. The Labsystems and Medac tests have the ability to diagnose acute C. trachomatis infection but lack the ability to reliably identify chronic infections. Furthermore, the Medac assay demonstrated low sensitivity and high cross-reactivity between chlamydial species (Bas et al, J Clin Microbiol 32:4082-4085, 2001). Therefore, incorporating C. trachomatis recombinant antigens in the Medac test failed to improve species specificity.

Numerous studies have demonstrated a correlation between antibody responses to chlamydial heat shock protein 60 (cHSP60) and pathologic sequelae in women (Brunham et al, J Infect Dis 165: 1076- 1081, 1992; Dieterle and Wollenhaupt, Hum Reprod 77:1352- 1356, 1996; Freidank et al, Eur J Clin Microbiol Infect Dis 75:685-688, 1997), including a significant association between the presence of antibodies to cHSP60 and PID (Eckert et al, J Infect Dis 775:1453-1458, 1997; Peeling et al, J Infect Dis 775:1153-1158, 1997; Witkin et al, Hum Reprod 75:1175-1179, 1998). An association of antibodies to cHSP60 and tubal factor infertility has also been reported (Arno et al, Fertil Steril 64:130-135, 1995; Claman et al, Fertil Steril 57:501-504, 1997; Freidank et al, 1997, supra). The identification of a C. trachomatis infection stage discriminator lead to the development of a commercial ELISA screening test based on cHSP60 (Medac, Hamburg, Germany). Investigators have subsequently evaluated the diagnostic potential of the cHSP60 ELISA test (Bax et al, Sex Transm Infect 50:415-416, 2004). Bax et al, demonstrated an 11% increased anti-cHSP60 antibody response in women with tubal pathology compared to women without tubal pathology (16%) and the control group comprising pregnant women (4.8%). In contrast, a more recent study showed an 8% decreased incidence of cHSP60 reactivity in the PID/Tubal Damage group compared to 28% in acute patients. Consequently, the ability of the cHSP60-based assay to distinguish various C. trachomatis infection stages may be limited. Whilst all these tests have the capacity to identify chlamydial infection, none can reliably differentiate between acute and chronic C. trachomatis infection.

There is a need, therefore, for improved diagnostic tests for Chlamydia, and especially for improved diagnostic tests which can differentiate different stages of a chlamydial infection. There is also a need to develop therapeutic protocols for the treatment of chlamydial infection. SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

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.

In one embodiment, therefore, 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. Although 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.

In another embodiment, therefore, the present invention provides a method for determining the presence or stage of chlamydial infection in a subject, said method comprising:

(i) obtaining a biological sample from a subject, said biological sample comprising antigen-binding molecules which are capable of forming complexes with one or more chlamydial antigens;

(ii) contacting the antigen-binding molecules with the one or more chlamydial antigens; and (iii) detecting the presence and/or amount of complexes formed between the antigen- binding molecules and the one or more chlamydial antigens

wherein the presence and/or amount of said complexes indicates the presence or stage of chlamydial infection in a subject.

In some embodiments of the present invention, 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. Examples of 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. A "homolog' includes a structurally or functionally similar protein in another strain or species of Chlamydia or in another genus of microorganism.

Hence, 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

(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 antibodies to one or more of said protein indicates the presence or stage of chlamydial infection in said subject.

In particular, 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:

(i) the presence of antibodies to any one of CT147, CT314, CT727, CT396, CT423, CTl 57 and/or CT413 is indicative of infection by a species of Chlamydia;

(ii) the presence of a greater level of antibodies to CT423 and CT396 compared to other proteins is indicative of acute infection by a sequence of Chlamydia; and

(iii) the presence of a greater level of antibodies to CTl 57 and CT727 and lower amounts of antibodies to CT423, compared to other proteins is indicative of chronic infection by a species of Chlamydia.

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.

Although 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. As such, 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.

Hence, 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. In another embodiment, therefore, 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.

In particular, 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.

Hence, 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.

Preferably the agent is an immunotherapeutic agent that is in the form of an immunotherapeutic composition such as, but not limited to, a vaccine. Accordingly, the present invention also provides for the use of the agent for the preparation of a composition. Hence, 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.

Hence, another aspect of the present invention provides a kit for identifying a chlamydial infection or for distinguishing between strains of Chlamydia said kit 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. In particularly preferred embodiments, the infection is caused by Chlamydia trachomatis. Furthermore, 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).

Table 1: Summary of Sequence Identifiers

BRIEF DESCRIPTION OF THE FIGURES

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. (Lanes: 1 = uninfected HEp-2 cells; 2 = HEp-2 cells infected with C. trachomatis L2; 3 = HEp-2 cells infected with C. trachomatis D; 4 = HEp-2 cells infected with C. trachomatis K; 5 = HE-2 cells infected with C. pneumoniae).

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. In the recovered acute stage, all antigens show a higher reactivity with ATPase displaying the greatest increase. In the chronic (PID) stage, antibody responses to CT 147 are reduced, and an equal reactivity of ATPase and

Hemolysin is observed. Across all infection phases, the 13.5kDa antigen exhibits moderate to high antigen reactivity.

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 5 is a photographic representation showing comparison of the western blot and coomassie-stained gel allowed correct protein identification (indicated by an arrow) and excision of the novel male marker for N-terminal sequencing. Indicated on the western blot are differential bands B and C. (I=C trachomatis L2 infected Hep-2 cells.)

Figure 6 is a photographic representation showing species and serovar comparison of novel 19kDa marker between the 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 the three bands previously observed in female patients. Boxed is the novel male marker. (Lanes: l=uninfected Hep-2 cells, 2=Hep-2 cells infected with C. trachomatis Ul, 3=Hep- 2 cells infected with C. trachomatis D, 4=Dep-2 cells infected with C. trachomatis K and 5-Hep-2 cells infected with C. pneumoniae).

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.

DETAILED DESCRIPTION OF THE INVENTION

Prior to describing the present invention in detail, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

It must also be noted that, as used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to 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.

In one embodiment, 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.

Reference herein to "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.

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.

In describing the present invention, various groups of subjects are tested. These groups are defined as follows:

(i) acute Chlamydia infection;

(ii) recovered acute Chlamydia infection;

(iii) chronic Chlamydia infection;

(iv) second acute Chlamydia infection (referred to herein as "204" group);

(v) pelvic inflammatory disease;

(vi) twelve months subsequent to reinfection (referred to herein as "212" group);

(vii) adult male control; and

(viii) child male control.' In one embodiment of the present invention, 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). One or two or three or four or five or six or all seven proteins may be used in a detection assay.

Hence, 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.

In particular, 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.

More particularly, 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:

(i) the presence of antibodies to any one of CT147, CT314, CT727, CT396, CT423, CTl 57 and/or CT413 is indicative of infection by a species of Chlamydia; (ii) the presence of a greater level of antibodies to CT423 and CT396 compared to other proteins is indicative of acute infection by a sequence of Chlamydia; and

(iii) the presence of a greater level of antibodies to CTl 57 and CT727 but lesser amounts of antibodies to CT423, compared to other proteins is indicative of chronic infection by a species of Chlamydia.

The term "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).

Reference herein to "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. Preferably, 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. In some embodiments of the present invention, 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).

Variant nucleic acid sequences 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. In accordance with the present invention, 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).

Terms used herein to describe sequence relationships between two or more nucleic acid sequences or protein sequences include "reference sequence", "comparison window",

"sequence identity", "percentage of sequence identity" and "substantial identity". A

"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. Reference also may be made to the BLAST family of programs as for example disclosed by 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.

The term "sequence identity" as used herein 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. Thus, 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. For the purposes of the present invention, "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.

Typically, 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.

Reference herein to "complementary" and "complementarity" refers to sequences of nucleotides related by the base-pairing rules. For example, the 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.

Alternatively, 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). In this regard, 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.

In relation to the blotting methods described above, 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. In dot blotting and slot blotting, 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.

Typically, the following general procedure can be used to determine hybridisation conditions. 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:5), CT396 (SEQ ID NO:7), CT423 (SEQ ID NO:9) and CT157 (SEQ ID NO:11), is labeled as described above, and the ability of this labeled nucleic acid sequence to hybridise with an immobilised nucleic acid sequence is analysed.

It will be understood that 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° C₅ and (i) 2xSSC, 0.1% w/v SDS; or (ii) 0.5% w/v BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 5% w/v SDS for washing at room temperature.

Suitably, 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% BSA₅ 1 mM EDTA, 0.5 M NaHPO₄ (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 BSA₅ 1 mM EDTA₅ 40 mM NaHPO₄ (pH 7.2), 5% w/v SDS for washing at 60-65° C

Preferably, 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° C₅ 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 EDTA₅ 0.5 M NaHPO₄ (pH 7.2), 7% w/v SDS for hybridisation at 65° C₅ and (i) 0.2 x SSC, 0.1% w/v SDS; or (ii) 0.5% w/v BSA, ImM EDTA, 40 mM NaHPO₄ (pH 7.2), 1% SDS for washing at a temperature in excess of 65° C.

Other stringent conditions are well known in the art. "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.

A skilled addressee will recognise that various factors can be manipulated to optimise the specificity of the hybridisation. Optimisation of the stringency of the final washes can serve to ensure a high degree of hybridisation. For detailed examples, see 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.

While stringent washes are typically carried out at temperatures from about 42° C to 68° C, one skilled in the art will appreciate that other temperatures may be suitable for stringent conditions. Maximum hybridisation rate typically occurs at about 20° C to 25° C below the T_m for formation of a DNA-DNA hybrid. It is well known in the art that the T_m is the melting temperature, or temperature at which two complementary polynucleotide sequences dissociate. Methods for estimating T_m are well known in the art (see Ausubel et al, "Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998.

In general, the 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)

wherein: 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;% formamide is the percent formamide concentration by volume; length is the number of base pairs in the DNA duplex.

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.

In a preferred hybridisation procedure, a membrane (e.g., a nitrocellulose membrane or a nylon membrane) containing 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. 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.

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. In some embodiments, 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). A double stranded region formed through the hybridization of an oligonucleotide (i.e., primer) to a single-stranded form of the target nucleic acid sequence is required to prime (start) the reaction.

By "primer" is meant 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. Alternatively, 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.

In other embodiments, 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. Alternatively, if 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.

The term "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. The term "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.

Variant polypeptides may be deduced from other species belonging to the family Chlamydiaceae by isolation of nucleic acid variants by standard protocols known in the art. In general, 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%.

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.

Reference herein to "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. The term "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, λ, α, γ (IgGi, IgG₂, IgG₃, IgG₄), δ, ε and μ 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. In addition to antibodies, 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. Methods of producing polyclonal antibodies are well known to those skilled in the art. Exemplary protocols which may be used are described for example in Coligan et al, "Current Protocols In Immunology", John Wiley & Sons Inc, 1991 and Ausubel et al, "Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998.

In lieu of the polyclonal antisera obtained in the production species, 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. Alternatively, 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. 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 Plϋnckthun et al, (In "Antibody engineering : A practical approach", 203-252: 1996).

Alternatively, 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. 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).

Also contemplated as 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).

Alternatively, the antigen-binding molecule may comprise a "minibody". In this regard, minibodies are small versions of whole antibodies, which encode in a single chain the essential elements of a whole antibody. Suitably, 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.

In an alternate embodiment, the antigen binding molecule may comprise 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. 57:1579-1584, 1992), or by use of domains (such as the leucine zippers jun and fos) that preferentially heterodimerise (Kostelny et al, J. Immunol. 148:1547-1553, 1992). In an alternate embodiment, the multivalent molecule may comprise a multivalent single chain antibody (multi-scFv) comprising at least two scFvs linked together by a peptide linker. In this regard, 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. Typically, 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. Hence, examples of biological samples include serum, whole blood, urine and secretions or washings.

For example, the biological sample may comprise fallopian tube washings from infertile women. The 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. Consistent with the present invention, the 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.

Any suitable technique for determining formation of an antigen-binding molecule- target antigen complex may be used. For example, an antigen-binding molecule according to the invention, having a reporter molecule associated therewith may be utilised in immunoassays. Such 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. For example, reference may be made to 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.

Two site assays are particularly favoured for use in the present invention. A number of variations of these assays exist, all of which are intended to be encompassed by the present invention. Briefly, in a typical forward assay, 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. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen complex, 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. Variations on the forward assay include 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. In accordance with the present invention, 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. From the foregoing, it will be appreciated that 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.

In the case of a direct visual label, use may be made of 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.

A large number of 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. 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. 5,227,487; 5,274,113; 5,405,975; 5,433,896; 5,442,045; 5,451,663; 5,453,517; 5,459,276; 5,516,864; 5,648,270 and 5,723,218. In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodates. As will be readily recognised, however, a wide variety of different conjugation techniques exist which are readily available to the skilled artisan. The substrates to be used with the specific enzymes are generally chosen for the production of, upon hydrolysis by the corresponding enzyme, a detectable colour change. Examples of suitable enzymes include those described supra. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, 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.

Alternately, fluorescent compounds, such as fluorescein, rhodamine and the lanthanide, europium (EU), may be chemically coupled to antibodies without altering their binding capacity. 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) are well established in the art. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules may also be employed.

It will be well understood that other means of testing target polypeptide (e.g., CT147,

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.

Alternatively to the methods discussed to this point, 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.

As noted hereinbefore, 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. In so far as obtaining antibodies from a subject for use which the diagnostic method of the present invention, preferably 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. In another embodiment, therefore, the present invention provides a method for determining the presence or stage of chlamydial infection in a subject, said method comprising:

(i) obtaining a biological sample from a subject, said biological sample comprising antigen-binding molecules which are capable of forming complexes with one or more chlamydial antigens; (ii) contacting the antigen-binding molecules with the one or more chlamydial antigens; and (iii) detecting the presence and/or amount of complexes formed between the antigen- binding molecules and the one or more chlamydial antigens

As used herein, "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. Preferably, the chlamydial antigens are derived from the organism that causes the chlamydial infection.

In particularly preferred embodiments of the present invention, the chlamydial antigens are selected from the group comprising CT147, CT314, CT727, CT396, CT423, CTl 57 and CT413. Hence, 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.

In embodiments relating to the detection of nucleic acids and 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.

In embodiments relating to the detection of antigen-binding molecules, such as a serological test kit, the kit of the present invention comprises one or more chlamydial antigens. In a particularly preferred embodiment, 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.

Hence, another aspect of the present invention provides a kit for identifying a chlamydial infection or for distinguishing between strains of Chlamydia said kit 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. For example, 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 recombinant neuclic acid; and (d) isolating the recombinant polypeptide. By "operably linked" is meant that transcriptional and translational regulatory nucleic acid sequences are positioned relative to a protein-encoding nucleic acid sequence in such a manner that the protein-encoding nucleic acid sequence is transcribed and translated.

What constitutes suitable variants of the nucleic acid sequences of the present invention may be determined by conventional techniques. For example, 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. Alternatively, 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.

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. By "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. Accordingly, 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. Alternatively, 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.

In a preferred embodiment, 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. The main advantage of fusion partners is that they assist identification and/or purification of said fusion polypeptide. In order to express said fusion polypeptide, it is necessary to ligate a polynucleotide according to the invention into the expression vector so that the translational reading frames of the fusion partner and the polynucleotide coincide. Well known examples of 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. For the purposes of fusion polypeptide purification by affinity chromatography, 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. In a preferred embodiment, 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). 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. Preferably, 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. Well known examples of epitope tags for which specific monoclonal antibodies are readily available include c-Myc, influenza virus, haemagglutinin and FLAG tags. In an especially preferred embodiment, 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. Alternatively, 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. Alternatively, 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. In particular, 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.

The 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.

In addition, 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.

Reference herein to "treatment" may mean a reduction in the severity of an existing condition. The term "treatment" is also taken to encompass "prophylactic treatment" to prevent the onset of a condition. The term "treatment" does not necessarily imply that a subject is treated until total recovery. Similarly, "prophylactic treatment" does not necessarily mean that the subject will not eventually contract a condition.

Preferably 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. Typically, 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. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, 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.

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. Useful carriers are well known in the art and 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. Alternatively, a fragment or epitope of a carrier protein or other immunogenic protein may be used. For example, a haptenic peptide can be coupled to a T cell epitope of a bacterial toxin, toxoid or CRM. In this regard, reference may be made to U.S. Patent No 5,785,973.

In addition, 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-D-isoglutamine (CGP 11637, referred to as nor-MDP), N- acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1 '-2'-dipalmitoyl-sn-glycero-3- hydroxyρhosphoryloxy)-ethylamine (CGP 1983 A, referred to as MTP-PE); dimethylglycine, tuftsin; oil emulsions; and mineral gels such as aluminum phosphate, aluminum hydroxide or alum; Freunds incomplete adjuvant, Freunds complete adjuvant, tetanus toxoid, diphtheria toxoid, ISCOMS, QuilA, and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% v/v squalene/Tween-80 emulsion. 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.

In some embodiments, the immunotherapeutic composition may be administered via a mucosal route such as, but not limited to, orally, urogenitally or transdermally or combination of these. Accordingly, the adjuvant is preferably a mucosal adjuvant.

Preferably, 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. The same techniques can be applied to other molecules with mucosal adjuvant or delivery properties such as E. coli heat labile toxin. Other compounds with 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. Alternatively, 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 "Animal Parasite Control Using Biotechnology", CRC Press, 1992).

The 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).

Ideally, the infectivity of the virus is destroyed without affecting the proteins that carry the immunogenicity of the virus. From the foregoing, it will be appreciated that 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).

Live vaccines are particularly advantageous because they lead to a prolonged stimulus that can confer substantially long-lasting immunity. Thus, as an alternative to the delivery of immunogenic agents in the form of a therapeutic or prophylactic immunotherapeutic composition, 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. In addition, epitopes of other pathogenic microorganisms can be incorporated into the compositions.

In one embodiment, this will involve the construction of a recombinant vaccinia virus to express a nucleic acid sequence according to the invention. Upon introduction into a host, the recombinant vaccinia virus expresses the immunogenic agent, and thereby elicits a host CTL response. For example, reference may be made to U.S. Patent No. 4,722,848, which 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.

In a further embodiment, 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. For example, 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). Thus, the invention also contemplates 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.

With regard to 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. In an alternate embodiment, 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. A discussion of 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. In particularly preferred embodiments of the present invention, 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.

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).

The present invention is further described by the following non-limiting examples.

EXAMPLE 1 Experimental Procedures

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).

Group 1 : Acute female patients diagnosed with first-time C. trachomatis infection estimated to have been acquired within the preceding 4 months (n = 9).

Group 2 : Recovered Acute female patients diagnosed with first-time C. trachomatis infection estimated to have been acquired more than 12 months previously (ranging from 12 months to 168 months previous (n = 8).

Group 3 : PID patients with presumptively diagnosed pelvic inflammatory disease based on clinical history, a confirmed medical history and serological evidence of previous C. trachomatis infection (n = 10).

Group 4 : 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).

Group 5 : Controls (uninfected adult females). Samples were obtained from patients attending the Brisbane Red Cross Blood Bank who were serologically negative for C. trachomatis. Negative Control patients, uninfected and C. trachomatis negative (n = 12). AIl patient serum samples were assayed for C. trachomatis IgG using commercial EIA (Labsystems Chlamydia trachomatis IgG EIA) with a 1/10 sera dilution as per manufacturer's instructions. A positive result was based on a ratio comparing sample absorbance and the designated cut-off value (<1.0 negative, <1.4 equivocal, <2.5 positive and >2.5 high positive). C. pneumoniae serology was determined using MIF (Focus Diagnostics (USA) Chlamydia MIF IgG) according to manufacturer's instructions. Positive titres for all patients ranged between 256 and 1024.

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% CO₂and 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. 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 -2O⁰C until required for use in SDS- PAGE/western blot experiments. Separate to the infected culture (I), HEp-2 only cells (UI) were maintained in 5% v/v FCS-DMEM, and proteins extracted as described above.

Identification of Reactive C. trachomatis Proteins in Patient Sera by Western Blotting:

In order to identify potential diagnostic 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, 2μL 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). Proteins were transferred at 4⁰C, 100 volts for 1 hour to Hybond C Extra nitrocellulose membranes (Amersham Biosciences) using 3-(cyclohexylamino)-l-propane sulphonic acid (CAPS) buffer and the membranes blocked (5% w/v skim milk powder in PBS containing 0.1% v/v Tween 20 (SM-PBS-T)) for 1 hour at RT with rocking. Patient sera from the five patient groups were diluted 1:1000 in SM-PBS-T and incubated with the membranes at RT for 1 hour with shaking. After washing with PBS-T (PBS, 0.1% v/v Tween 20), 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).

Immunoprecipitation of Chlamydia Specific Antibodies: Twenty-four hour C. trachomatis L2 infected HEp-2 cell monolayers were washed in cold PBS, trypsinised and lysed with 5mL 1% v/v TritonXIOO. A ImL aliquot of the cell lysate was centrifuged at 1200Og for 10 minutes at 4°C. The supernatant was mixed with 20μL of patient sera and incubated at 4°C for 4 hours with rotation. During the lysate/antibody incubation, protein G attached to micro beads (Amersham Biosciences) was prepared as per manufacturer's instructions. The coupled lysate/antibody mix was directly added to 200μL 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 50μL of 2X sample buffer giving a final volume of ~100μL. 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:

Proteins precipitated by the protein G beads were separated using a 12.5% w/v polyacrylamide SDS-PAGE gel and transferred at 4⁰C, in CAPs buffer, to immobilon-P Polyvinylidene Fluoride (PVDF) membrane prepared as per manufacturer's instructions (Amersham Biosciences). 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 chlamydial antigens of interest.

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 β-lactoglobulin standard was used to verify sequencer performance.

Antigenic Target Identification and Verification via Mass Spectrometry:

Identification of bands B and C was determined by N-terminal sequencing. MS was used to identify band A and to confirm the identity of bands B and C. No identification of band D using either method was possible due to the small amount of protein. NU-PAGE 4%- 12% Bis-Tris 1 well SDS-PAGE gels (Invitrogen) were used to separate lOOμL of extracted chlamydial protein in IX MOPS buffer (5OmM 3 N-morpholino propane sulfonic acid, 3.5mM SDS, 5OmM EDTA, pH 8.0) at 4⁰C, 120 volts for 4 hours. A small segment of gel containing a section of protein was transferred in CAPs buffer to Hybond C Extra nitrocellulose membrane (Amersham Biosciences) and reserved for standard western blotting. The remaining gel was Coomassie stained for 30 minutes and destained (40% v/v methanol and 10% v/v glacial acetic acid) until background was removed. The coomassie stained gel and western blot were aligned to allow excision of differential chlamydial antigens of interest. In preparation for MS identification, bands were excised, dried and in- gel digested, reduced and alkylatated. Briefly, 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. To each gel piece, 200μL of 20OmM NH₄HCO₃/50% v/v ACN were added then incubated at 37°C for 45 minutes and supernatant discarded (X2). Destained gel pieces were dried in Speedvac (Savant) on low heat for 1 hour. For reduction, 100 μL of 2OmM dithiotreitol (DTT) in 25mM NH₄HCO₃ was added and incubated for 1 hour at 65°C and then the supernatant discarded. To alkylate protein samples, lOOμL of 5OmM iodoacetamide (IAA) in 25mM NH₄HCO₃ was added and incubated in the dark for 40 minutes at 37⁰C. Gel pieces were then washed twice in 200 μL 25mM NH₄HCO₃ for 15 minutes at 37°C, with supernatant discarded each time. Samples were reduced and gel fragments placed in Speedvac (Savant) for 1 hour. Gel pieces were rehydrated by the addition of 20μL 25mM NH₄HCO₃, pH 8, containing 0.02μg/μL trypsin and incubated at RT for 1 hour. An additional 50μL 25mM NH₄HCO₃ in 50% ACN was added to each gel slice and incubated at 37°C for 18 hours. To recover peptides, the tryptic supernatant was collected and 50μL of 0.1% TFA was added to each gel slice and incubated for 45 minutes at 37°C. The supernatant was again collected and pooled with the original decanted supernatant and washed twice with TFA. Peptides were concentrated via zip tipping, spotted onto a MS plate and analysed. Fragment patterns were matched to potential proteins using Mascot software.

Species and Serovar Specificity of the Identified Novel Markers: To determine the species and serovar specificity of our identified antigenic targets, a small subset of patients (n = 3/group) were screened via SDS-P AGE/western blot against C. trachomatis serovars L2, D, K, and C. pneumoniae. All C. trachomatis and C. pneumoniae infections, protein extractions and western blots were performed as described in section A of the materials and methods section. Precast SDS-PAGE 4%-20% gradients gels (BioRad) were used for this comparative study. EXAMPLE 2 Identification of Novel Diagnostic Markers by Western Blotting with Patient Sera

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. trachomatis negative by PCR, also demonstrated reactivity to this antigen. Band B was shown to be reactive to 80% and 100% of PID and acute multiply infected patients respectively. In contrast, both the acute and recovered acute groups demonstrated a decreased reactivity of 38%. Acute multiply infected and PID patients that were reactive to band B also recognised band C. Antibody reactivities of the acute and recovered acute groups to band C were 54% and 50% respectively. In addition, 8% of the negative control group also displayed reactivity to band C. All PID patients were reactive to Band D. Furthermore, 54%, 50% and 100% of the acute, recovered acute and acute multiply infected groups respectively also showed antibody reactivity.

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.

DIFFERENTIAL BANDS EIA MIF

PATIENT GROUPS ( :%)

A B C D

C tr C pn

Estimated Band Size (kDa) >113 72.4 44.6 13.5

Acute (H=I 3) 46 38 54 77 62 54

Recovered Acute (n=8) 63 38 50 50 63 50

Acute Multiply Infected («=3) 67 100 100 67 100 25

PID (K=IO) 10 80 80 100 80 60

Negative Control («=40) 2 8 8 30 0 45

EXAMPLE 3 Identification of the Differential Chlamydial Antigens

Band A could not be identified via N-terminal sequencing as the protein was not successfully precipitated from the C. trachomatis host-cell lysate. However, 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). The N-terminal sequence of band B showed 5/7 amino acid matches with CT727 (Metal transport P-type ATPase - 70.5kDa) in BLAST searches of the Chlamydia microbial (TIGR) database. However, MS analysis of band B suggested a match to CT396 (HSP70 - 70.8kDa). Homology searches of the N-terminal sequence of band C suggested two possible protein candidate matches: CTl 57 (Phospholipase D Endonuclease - 45.3kDa), and CT423 (probable Hemolysin containing a cystathionine beta synthase domain (CBS) - 41.5kDa) with both proteins matching 6/7 amino acids. Band D could not be identified by either method due to the limited amount of protein isolated from the SDS-PAGE gels.

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.

BAND GENE FUNCTION MW (kDa)

Band A CT147 Hypothetical Protein 162.1

CT314 DNA-directed RNA polymerase 154.9

Band B CT727 Metal transport P-type ATPase 70.5 CT396 Heat Shock Protein 70 71.1

Band C CT157 Phospholipase D Endonuclease 45.3 CT423 Probable Hemolysin 41.6 EXAMPLE 4

Species and Serovar Specificity of Marker Bands

Species and serovar specificity of the four bands A, B, C and D were investigated by individually reacting sera from five patients in acute, recovered acute, PID and negative control groups against preparations of C. trachomatis Ul, C. trachomatis D, C. trachomatis K and C. pneumoniae (Figure 2). Results from acute, recovered acute and PID patients were combined and analysed for specificity (Table 4). Bands A, B, C and D were observed in C. pneumoniae and all C. trachomatis serovars screened, consequently no serovar or species band specificity was detected. C. trachomatis serovar D exhibited the lowest combined specificity of 22% to the four chlamydial antigens. In contrast, C. 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.

Table 4. Five serum samples from each acute, recovered acute and PID groups were analysed to determine species and serovar specificity of the four identified bands in C. trachomatis L2, C. trachomatis D, C. trachomatis K and C. pneumoniae. Negative control patients were also screened («=5).

Percentage (%) Presence in

Band C pn

L2 D K

A 47 27 33 40

B 53 40 46 33

C 80 20 20 47

D 73 0 27 53 EXAMPLE 5 Diagnostic Potential of the Antigens

For the purpose of developing a serological diagnostic test capable of discrimination between acute and chronic C. trachomatis infection, we combined the identified antigens into panels and evaluated their predictive potential (Table 5). Antibody response to antigens A or B or C demonstrated the greatest sensitivity (75%) and specificity (74%) compared to other potential formats. As a consequence, panel 3 with a positive predictive value (PPV) of 58% and a negative predictive value (NPV) of 86% is the preferred diagnostic format for acute infection. In contrast, other panel formats such as 1 and 2 have significantly reduced sensitivity (41% and 50%) respectively, but higher specificity (96%).

The 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

80% sensitivity and 85% specificity for diagnosis of chronic infection.

Table 5. Sensitivity and specificity values of the various test formats using antigens A, B, C and D for diagnosis of acute and chronic chlamydial infection. (Sens = Sensitivity, Spec = Specificity, PPV = Positive Predictive value, NPV = Negative

Predictive Value, + = both antigens must be positive).

INFECTION STAGE _{p τ} ^ * _{ppv Npy}

1. A +B or C 33 96 80 75

2. C + A orB 42 96 83 77

Acute and Recovered 3. Acute Patients A orB or C 75 74 58 86

4. A or B or C orD 79 56 46 85

5. B or C orD 75 56 45 82

PID Patients 6. B + C 80 86 47 97 In essence, the antibodies to the antigens detect infection or stage of infection such that:

(i) the presence of antibodies to any one of CT147, CT314, CT727, CT396, CT423, CT157 and/or CT413 is indicative of infection by a species of Chlamydia;

EXAMPLE 6 Temporal Response of Identified Antigens during C. trachomatis Infection Stages

The data were used to analyse and potentially predict the temporal response of the four bands during acute, recovered acute, acute multiply infected and PID C. trachomatis infection stages (Figure 3). The acute phase of infection demonstrates moderate-high antibody reactivity to all bands. In the recovery phase of infection, reactivity to bands A and B are maintained whilst a reduction to the remaining bands is observed. In contrast, acute patients multiply infected exhibit a dramatic increase of antibody recognition to bands B, C and D. Furthermore, antigen reactivity to bands B and C exceeds the singly infected acute group by 62% and 38%, respectively. During the acute, recovered acute and acute multiply infected stages antibody reactivity to band A is constant however, chronic infection sees a dramatic decrease to only 10%. EXAMPLE 7 Identification of Novel Diagnostic Marker by Western Blotting

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. pneumoniae antibodies in the acute, recovered acute and multiply infected patients. EIA testing showed an 18% incidence in the recovered acute group, but moderate to high levels in the acute, 204 and 212 patient groups. Although the chronic patient was diagnosed C. trachomatis positive via PCR, serology did not detect C. trachomatis antibodies. Table 6. Prevalence and estimated molecular weights of the four differential bands with positive C. trachomatis and C. pneumoniae serology witnessed for each patient group.

DIFFERENTIAL BANDS

EIA/MIF (%)

PATIENT GROUPS (%)

B C D M

C. tr C. pn

Estimated Band Size (kDa) 72.4 44.6 13.5 19

Acute («=5) 100 100 80 80 40 60

Recovered Acute («=11) 55 36 64 82 18 82

204 («=1) 100 100 100 100 100 100

212 («=2) 50 50 0 50 50 50

Chronic («=1) 100 100 0 0 0 0

Adult Male Control («=4) 50 50 25 25 NA NA

NA, not available

EXAMPLE 8 Novel Marker Identification via N-terminal Sequencing

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. The 16 BLAST results demonstrated absolute homology (E value = 21.8, bits = 138) between the identified amino acids and numerous genera, and identified C. trachomatis Probable Outer Membrane Protein B (PmpB) as the novel gene candidate. Further investigation showed PmpB to have a molecular weight of 181. SkDa, approximately 9.5 times greater than the anticipated protein size. Original C. trachomatis culture preparations (I) 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.

Table 7. NCBI BLASTP alignments of the most significant N-terminal sequence matches. Highlighted is the identified C. trachomatis target, Probable Outer

Membrane Protein B (PmpB).

Score E

Sequences producing significant alignments : (Bits) Value qi|2506946|sp|P16429|HYCC ECOLI Formate hydrogenlyase subunit... 21.8 138 gi|20138462|splQ9WVE91ITSNl RAT Intersectin-1 (EH domain and ... 21.8 138 qil5902737|sp|Q99128|APlGl USTMA AP-I complex subunit gamma-1... 21.8 138 qi|124142|sp|P28925|ICP4 EHVlB Trans-acting transcriptional p... 21.8 138 gi|39931305|sp|Q9RTG5|IF2 DEIRA Translation initiation factor IF 21.8 138 gi 11710593 |sp|P51408|RLA2 TRYBB 60S acidic ribosomal protein P2 21.8 138 qi|124143|sp|P17473|ICP4 EHVlK Trans-acting transcriptional p... 21.8 138 qil267469|sp|P29941|YCB8 PSEDE Hypothetical 19.2 kDa protein in 21.8 138 gi|118353|sp|P13187|DCOA KLEPN Oxaloacetate decarboxylase alpha 21.8 138 gi|59797779|sp|Q6S6ϋ0|ICP4 EHVlV Trans-acting transcriptional ... 21.8 138 gi I 22096375 jsplQ9V7H4 I RWl DROME RWl protein homolog 21.8 138 gi 138257667 |sp|Q8PEH5 I DNAA XANCP Chromosomal replication initiat 21.8 138 qi I 81779155 I sp|Q98EV7 IATPG RHILO ATP synthase gamma chain (ATP S 21.8 138 gi|14195035|sp|O84418|PMPB CHLTR Probable outer membrane protein 21.8 138 gi I 67460982|sp|O60346|PHLPP HUMAN PH domain leucine-rich repe... 21.8 138 gi|68565584|sp|Q6T2641MAMLl MOOSE Mastermind-like protein 1 (Mam 21.8 138

EXAMPLE 9

Species and Serovar Specificity of Novel Male Marker

Species and serovar specificity of the novel male marker was determined by probing C. trachomatis L2, D and K₅ 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%). Moderate cross-reactivity was observed for band C in C. trachomatis serovar L2 and C. pneumoniae. In contrast, specificity of C. trachomatis serovars D and K was markedly reduced to 5% and 15%, respectively. Band D was shown to be poorly reactive across all tested chlamydial species and serovars. Of the four bands analysed for species and serovar specificity, band M demonstrated the highest overall cross-reactivity. C. trachomatis L2 and C. pneumoniae showed an 80% and 75% specificity, respectively, whilst both remaining C. trachomatis serovars exhibited a lower cross-reactivity of 35%.

Table 8. In silico tryptic digest of PmpB by Peptide Mass Program, ExPASy

(www.expasy.org). Highlighted are the identified N-terminal sequence amino acids contained within the protein fragment. Bolded and underlined is the conserved and highly repetitive amino acid motif common to the Pmp family.

EXAMPLE 10 Temporal Response of Novel Marker during C. trachomatis Infection Stages

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. In the recovery phase of infection, 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. Furthermore, 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

C.

Table 9. All serum samples from Acute, Recovered Acute, 204, 212 and Chronic patients were probed against C. trachomatis L2, C. trachomatis D, C. trachomatis K and C. pneumoniae to determine male species and serovar specificity of bands B, C, D and M.

Percentage (%) Presence in

Band C. tr C pn

L2 D K

B 70 15 45 60

C 60 5 15 45

D 55 0 15 20

M 80 35 35 75 Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

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Claims

CLAIMS:

1. 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), CT157 (SEQ ID NO:12) and CT413 (SEQ ID NO: 13) wherein:

(iii) the presence of a greater level of antibodies to CTl 57 and CT727 but low or absence of antibodies to CT423, compared to other proteins is indicative of chronic infection by a species of Chlamydia.

2. The method of Claim 1 wherein the subject is a human.

3. The method of Claim 1 or 2 wherein the biological sample is serum, whole blood, urine or a secretion.

4. The method of Claim 2 wherein the acute stage of chlamydial infection is selected from acute, recovered acute, acute multiply infected or pelvic inflammatory disease (PID).

5. The method of Claim 2 wherein the species of Chlamydia detected is Chlamydia trachomatis or Chlamydia pneumoniae.

6. The method of Claim 2 wherein the species of Chlamydia is Chlamydia trachomatis.

7. The method of Claim 6 wherein the Chlamydia trachomatis is strain C, D, K or Ul.

8. A kit for identifying a chlamydial infection or for distinguishing between strains of Chlamydia said kit comprising a support or container adapted to contain one or more proteins selected from CT147, CT314, CT727, CT396, CT423, CT157 and CT413, said support or container capable of receiving a biological sample potentially comprising antibodies to one or more of said proteins.

9. The kit of Claim 8 comprising at least two of said proteins.

10. The kit of Claim 8 comprising at least three of said proteins.

11. The kit of Claim 8 comprising at least four of said proteins.

12. The kit of Claim 8 comprising at least five of said proteins.

13. The kit of Claim 8 comprising at least six of said proteins.

14. The kit of Claim 8 comprising all seven proteins.

15. A vaccine against chalymidal infection said vaccine comprising at least one protein selected from CT147, CT314, CT727, CT396, CT423, CT153 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.

16. 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.

17. The method of Claim 16 wherein the subject is a human.

18. Use of a protein selected from CT147, CT314, CT727, CT396, CT423, CTl 57 and CT413 in the manufacture of a diagnostic agent for chlamydial infection.

19. Use of a protein selected from CT147, CT314, CT727, CT396, CT423, CTl 57 and CT413 in the manufacture of a medicament to treat chlamydial infection.

20. An isolated protein selected from the list consisting of CT147, CT314, CT727, CT396, CT423, CT157 and CT413.

21. An isolated antibody to the protein of Claim 20.