WO2014078890A1

WO2014078890A1 - Chlamydia trachomatis diagnostic peptide and method

Info

Publication number: WO2014078890A1
Application number: PCT/AU2013/001333
Authority: WO
Inventors: Wilhelmina May HUSTON; Shruti MENON
Original assignee: Queensland University Of Technology
Priority date: 2012-11-20
Filing date: 2013-11-20
Publication date: 2014-05-30

Abstract

An isolated peptide consisting essentially of the amino acid sequence ADTRGILWAVE or a variant or fragment thereof is provided as an indicator of whether a patient has been, or is, infected with Chlamydia. This peptide may be used alone of together with other Chlamydia-derived peptides to detect the presence of specific antibodies in patient sera. More particularly, the peptides may be recognized by serum antibodies of women who have previously been exposed to Chlamydia which is highly specific for detection of women with Chlamydia sequelae such as tubal infertility, pelvic inflammatory disease or ectopic pregnancy

Description

TITLE

CHLAMYDIA TRACHOMATIS DIAGNOSTIC PEPTIDE AND METHOD

TECHNICAL FIELD

THIS INVENTION relates to molecular diagnostics. More particularly, this invention relates to a peptide for detecting serum antibodies to Chlamydia trachomatis which may indicate a predisposition to post-infection sequelae, such as pelvic inflammatory disease, infertility, and ectopic pregnancy in women.

BACKGROUND

Chlamydia (C.) trachomatis is the most prevalent sexually transmitted bacterial infection worldwide, with 1 ,307,893 infections reported in the USA in 2010 [1]. The infection is frequently asymptomatic which means that the actual case numbers are likely to be far higher than the notifications [2-3]. The major burden of Chlamydia infections relates to the serious sequelae, such as pelvic inflammatory disease, infertility, and ectopic pregnancy in women. The asymptomatic nature of the infection and the detection of the sequelae much later in life mean that it is extremely difficult to predict the number of untreated infections which progress to serious sequelae.

A retrospective study of 43,715 women in Sweden over a 10-15 year time frame, using health data repositories, indicated that for the women who tested positive to C. trachomatis infection; 5.6% had pelvic inflammatory disease (PID), 2.7% had ectopic pregnancy, and 6.7% had infertility [4]. A review of published studies by Land and co-workers found that diagnosed lower genital tract infection can progress to PID in 0.43-31% of cases, to tubal infertility in 0.02-4.6% of cases, and PID progresses to tubal infertility in 1 1.4-20% of cases [5].

The pathogenic mechanism, host genetic factors, and other epidemiological factors which lead to serious sequelae in a proportion of infected women are not well understood, although it is clear that infertility in women is associated with tubal damage resulting in fallopian tube occlusion. Additionally, minor tubal adhesions or damage to the cilia lining the fallopian tubes are also considered to be C. trachomatis infection related sequelae which impact on fertility. This has been further supported

Substitute Sheet

(Rule 26) RO/AU by a recent finding by Coppus and co-workers that participants attending a fertility clinic with high C. trachomatis serum antibody titers by MIF or ELISA (>1 :32 or 1.1 respectively) with patent tubes by HSG or laparoscopy, had a 33% lower ongoing pregnancy rate [6].

C. trachomatis antibody titers (CAT) are currently the gold standard for diagnosis of Chlamydia related infertility in women. There are two main types of tests which most commonly used; immunofluorescence tests (MIF, IF, or WIF) or ELISA. There are several different commercially available ELISA tests, and they are typically based on major outer membrane protein (MOMP) peptides.

Unfortunately, there has been widely reported variation in the sensitivity and specificity of these tests for the diagnosis of tubal infertility between different studies. A recent metaanalysis by Broeze and colleagues confirmed this considerable variation even when the same commercial tests were used between different studies. The ELISA (mainly Medac, Biomerieux, Labsystems) sensitivity varied from 12- 69%, and specificity varied from 62-100% [7]. The immunofluorescence methods were more sensitive (50-91%) but had lower specificity (35-85%) [7],

There are also differences in clinical practice regarding the use CAT as a routine part of infertility work up. Given the high level of variability in the test's performance, and that there are other causes of tubal infertility, many clinics prefer to use hysterosalpingography or laparoscopy to diagnose tubal infertility in women and do not use CAT at all. The guidelines regarding use of CAT in the preliminary infertility work up are also highly variable. The Dutch Society for Obstetrics and Gynecology recommends CAT during initial fertility work up and suggest a fixed cut off of IgG MIF 1 :32, or ELISA>1.1 as positive cut off for CAT[8], whereas CAT is not mentioned in Australian diagnosis recommendations.

Despite these differences in clinical acceptance, it remains possible that new assays targeting different chlamydial antigens could be used to develop a more sensitive and specific CAT to predict female infertility due to C. trachomatis past infection which could be implemented effectively at IVF clinics during the preliminary work up. A highly specific CAT could potentially also be used by women in a primary care setting in order to advise of their likely fertility status to enable family planning and appropriate patient management. These applications of a new CAT assay have led to proteomics investigations within the field aiming to identify suitable chlamydial antigens which could be used to develop a more sensitive and specific CAT to predict Chlamydia-related infertility in women.

A genome wide study using an expression array identified that a combination of CT443 (OmcB) and CT381 (ArtJ) had a 67.5% sensitivity and 100% specificity [9]. A proteomic approach searching for B and T cell antigens recognized by patients with a high MIF titer frequently identified MOMP, HtrA, OmcB, TART, GroEL, LCR-E, and CT662 [10]. In another study using a western blot strategy on 2-D gels of the Chlamydia proteome MOMP, PmpD, and OmcB were found to be the most specific to infection positive participants (not solely infertility) [11]. HtrA as a full length protein was found to be immunoreactive from C. trachomatis infected participants [12].

SUMMARY

The present inventors have used a bioinformatics approach to predict antibody epitopes with unique sequence specificity to C. trachomatis from a selected subset of C. trachomatis antigens. Surprisingly, a peptide of the HtrA protein of C. trachomatis has led to the development of a diagnostic a test which is highly specific for diagnosis of Chlamydia-telated infertility in women.

In one aspect, the invention provides an isolated peptide comprising, consisting essentially of, or consisting of, the amino acid sequence

ADTRGILWAVE (SEQ ID NO: 1 ).

This aspect also provides a fragment of the isolated peptide comprising consisting essentially of, or consisting of, the amino acid sequence

ADTRGILWAVE (SEQ ID NO:l). In certain embodiments the fragment comprises, consists essentially of, or consists of, an amino acid sequence selected from the group consisting of: ADTRGILW (SEQ ID NO: 2); ADTRGIL (SEQ ID

NO:3) and TRGILVV (SEQ ID NO:4).

This aspect also provides a variant of the isolated peptide comprising, consisting essentially of, or consisting of, the amino acid sequence ADTRGILWAVE (SEQ ID NO: 1). In certain embodiments the variant comprises, consists essentially of, or consists of, an amino acid sequence selected from the group consisting of: GILVVAVEAGSP (SEQ ID NO:5) and ILWAVEAGSPA

(SEQ ID NO:6). This aspect of the invention also provides an isolated peptide comprising, consisting essentially of, or consisting of, an amino acid sequence set forth in any one of SEQ ID NOS:7-16, or a fragment or variant thereof.

This aspect also provides an isolated protein comprising one or more isolated peptides, fragments and/or variants of this aspect, wherein the isolated protein does not comprise a full length HtrA amino acid sequence, such as set forth in SEQ ID NO:17.

This aspect also provides an isolated protein comprising one or more isolated peptides and/or variants of this aspect, wherein the isolated protein further comprises a heterologous amino acid sequence.

In one embodiment, the isolated protein further comprises one or more amino acid sequences of SEQ ID NO.7-16, or a fragment thereof.

A further embodiment provides a fragment of the isolated peptide, variant or the isolated protein of this aspect.

In another aspect, the invention provides a method of determining whether a subject has been exposed to, or infected by, C. trachomatis, said method including the step of determining whether a biological sample obtained from a subject comprises an antibody that binds one or more isolated peptdies or isolated proteins comprising the amino acid sequence ADTRGILWAVE (SEQ ID NO:l), or a fragment or variant thereof, wherein if the biological sample comprises the antibody the subject has been exposed to, or infected by, C. trachomatis.

In some embodiments, the isolated peptide comprising the amino acid sequence of SEQ ID NO:l, or a fragment or variant thereof, may be used in combination with one or a plurality of other peptides, or a fragment or variant thereof. In particular embodiments, the other peptides respectively comprise the amino acid sequences of SEQ ID NO: 7- 16, or a fragment thereof.

In yet another aspect, the invention provides a method of determining whether a subject has, or is predisposed to, one or more C. trachomatis sequelae and/or a chronic G trachomatis infection, said method including the step of determining whether a biological sample obtained from a subject comprises an antibody that binds one or more isolated peptides or isolated proteins comprising the amino acid sequence ADTRGILWAVE (SEQ ID NO:l), or a fragment or variant thereof, wherein if the biological sample comprises the antibody the subject has, or is predisposed to, the one or more C. trachomatis sequelae and/or chronic C. trachomatis infection.

Suitably, the method is capable of distinguishing a predisposition to one or more C. trachomatis sequelae and/or a chronic infection from an acute infection with or by C. trachomatis.

In particular embodiments, the C. trachomatis sequelae include tubal factor infertility, ectopic pregnancy and pelvic inflammatory disease (PID).

In some embodiments, the isolated peptide comprising the amino acid sequence SEQ ID NO:l or a fragment or variant thereof, may be used in combination with one or a plurality of other peptides, or a fragment thereof. In particular embodiments, the other peptides respectively comprise the amino acid sequences of SEQ ID NO:7-l 6, or a fragment thereof.

In a further aspect, the invention provides an isolated antibody which binds an isolated peptide comprising the amino acid sequence ADTRGILVVAVE (SEQ ID NO: 1), or a fragment or variant thereof.

In one embodiment, the isolated antibody is a monoclonal antibody.

In a still further aspect, the invention provides a composition or kit comprising an isolated peptide comprising the amino acid sequence ADTRGILVVAVE (SEQ ID NO: 1 ), or a fragment or variant thereof.

Suitably, the composition or kit comprises one or more reagents that facilitate detection of antibodies that bind said peptide.

A yet further aspect of the invention provides an isolated nucleic acid comprising a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO:l), or a fragment or variant thereof. In some embodiments, the isolated nucleic acid may encode the isolated protein disclosed herein.

One embodiment of this aspect provides a genetic construct comprising the isolated nucleic acid in combination with one ore more other nucleotide sequences.

Another embodiment of this aspect provides a host cell comprising the genetic construct.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Receiver operator characteristic curve for the sera dilution of 1/200 comparing participants from sequelae and negative infection cohorts. In the box (bottom left) area under the curve (AUC) for 1/200, 1/400 and 1/800 sera dilutions are shown with calculated absorbance (450nm) thresholds for a 95% specificity cutoff. Dashed line indicates the 95% specificity (xaxis) cutoff which corresponds to 46% sensitivity (y-axis).

Figure 2: Peptide 11 (SEQ ID NO:l) was used in combination with peptides cHsp60-e2 (SEQ ID NO:7) and 443-n2 (SEQ ID NO: 10) in an ELISA to determine whether there were differences between acute infection and sequelae. Left three columns patient sera were used at 1 :200. Two different peptide concentrations were tested by diluting from the 1.5mg/ml stock solution: 1:100 dilution; right three columns at 1:200 dilution.

Figure 3: The peptide combinations (SEQ ID NOS: 1, 7 and 10) were used in an ELISA to evaluate the difference in the IgG subclasses (IgGj, IgG2, IgG₃ & IgG ) present in patient sera to determine whether there were differences between acute infection and sequelae. Patient sera were used at 1 :200 dilution. The peptides were tested at 1:100 dilution and at 1 :200 dilution from the 1.5 mg ml stock. Anti-Ig class- specific antibodies were used at 1:150 dilution.

DETAILED DESCRIPTION

The present invention has arisen, at least in part, from the discovery that a peptide ADTRGILVV (SEQ ID NO:2) contained within an amino acid sequence ADTRGILVV AVE (SEQ ID NO:l) of the HtrA protein of C. trachomatis has facilitated the development of a diagnostic a test which is highly specific for diagnosis of Chlamydia-related infertility in women. More particularly, this peptide is recognized by serum antibodies of women who have previously been exposed to C. trachomatis, which is highly specific for detection of women with C. trachomatis sequelae such as tubal infertility, PID or ectopic pregnancy. The peptide bound serum antibodies from women that had been infected with C. trachomatis compared to infertile women with no history of C. trachomatis infection or tubal damage. It is also proposed that this peptide could facilitate distinguishing between women that have had a single, treated infection versus women having multiple infections. Accordingly, an aspect of the invention provides a diagnostic test for predicting the risk of C. trachomatis sequelae such as tubal infertility, PID or ectopic pregnancy in "at risk" patients and more specifically in screening for potential infertility problems in rVF patients. The terms 'comprises', 'comprising' , 'includes', 'including' , or similar terms are intended to mean a non-exclusive inclusion, such that a method, system or apparatus that comprises a list of elements does not include those elements solely, but may well include other elements not listed.

By "consists essentially of is means that a peptide comprises a recited amino acid sequence and 1, 2 or 3 additional amino acids at the N- and/or C-terminus of the amino acid sequence.

A "protein" is an amino acid polymer comprising natural or non-natural D- or L-amino acids, as well understood in the art. A "peptide" is a protein consisting of fifty (50) or fewer amino acids.

A peptide "fragment" comprises at least 4, 5, 6, 7, 8, 9 or 10 amino acids of the entire amino acid sequence of a peptide. Suitably, the fragment is capable of binding or being recognized by an antibody which binds or recognizes the peptide.

An "antibody" is an immunoglobulin protein that may be polyclonal or monoclonal, inclusive of antibody fragments and includes recombinant antibodies and fragments thereof, Fab and Fab'2 fragments, diabodies and single chain antibody fragments {e.g. scVs), although without limitation thereto. Typically, an antibody present in biological sample, such as a serum sample, will be a polyclonal antibody.

One aspect of the invention provides an isolated peptide comprising, consisting essentially of or consisting of the amino acid sequence or ADTRGILWAVE (SEQ ID NO:l), or a fragment or variant thereof.

The peptide comprising, consisting essentially of or consisting of the amino acid sequence ADTRGILWAVE (SEQ ID NO:l) is derivable or obtainable from, or otherwise corresponds to, a fragment of a HtrA protein of C. trachomatis. The HtrA protein is also known as CT823 or DEGP, the amino acid sequence of which is set forth in SEQ ID NO: 17. The peptide sequence ADTRGILWAVE (SEQ ID NO:l is underlined in SEQ ID NO: 17: mkrllcvlls tsvfsspmlg ysaskkdska diclavssgd qevsqedllk evsrgfsrva akatpgwyi enfpktgnqa iaspgnkrgf qenpfdyfnd effnrffglp syreqqrpqq rdavrgtgfi vsedgywtn hhwedagki hvtlhdgqky takivgldpk tdlavikiqa eklpfltfgn sdqlqigdwa iaignpfglq atvtvgwsa kgrnqlhivd fedfiqtdaa inpgnsggpl lningqvigv ntaivsgsgg yigigfaips lmakrvidql isdgqvtrgf lgvtlqpids elatcyklek vygalvtdw kgspaekagl rqedvivayn gkeveslsal rnaislmmpg trvilkivre gktieipvtv tqiptedgvs alqkmgvrvq nitpeickkl glaadtrgil waveagspa asagvapgql ilavnrqrva sveelnqvlk nskgenvllm vsqgdwrfi vlksde

(SEQ ID NO:17) Although not wishing to be bound by any particular theory, it is proposed that the amino acid sequence ADTRGILW (SEQ ID NO: 2) is a putative B cell "epitope fragmenf of SEQ ID NO:l that is capable of being bound, recognized or detected by an antibody. Other putative epitope fragments of SEQ ID NO:l include ADTRGIL (SEQ ID NO:3) and TRGILW (SEQ ID NO:4), which are also fragments of SEQ ID NO:2.

Also provided are variants of the amino acid sequence of any one of SEQ ID NOS:l-16. Variants suitably comprise one or more conservative amino acid sequence substitutions, deletions or additions which do not substantially affect the binding of antibodies that bind the amino acid sequence set forth in any one of SEQ ID NOS: 1-16. In some embodiments, the variant has one, two, three, four or five conservative amino acid sequence substitutions, deletions and/or additions which do not substantially affect the binding of antibodies that bind the amino acid sequence set forth in any one of SEQ ID NOS: 1-16. In other embodiments, the variant has an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOS: 1 - 16.

Particular embodiments of variants of SEQ ID NO: 1 include a peptide comprising the amino acid sequence GILWAVEAGSP (SEQ ID NO:5) or ILWAVEAGSPA (SEQ ID NO:6).

The term "sequence identity" is used herein in its broadest sense to include the number of exact amino acid matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison. Sequence identity may be determined using computer algorithms such as GAP, BESTFIT, FASTA and the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res. 25 3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995-1999).

Also provided is an isolated protein comprising an amino acid sequence of a fragment of a HtrA/ CT823/DEGP protein. In certain embodiments, the isolated , protein comprises at least 12, 15, 18, 20, 25, 30, 40, 50, 60, 70, 80, 100, 120, 150,

200, 300, 350, 400, 450 or up to 480 contiguous amino acids of SEQ ID NO: 17 that comprises the amino acid sequence ADTRGILWAVE (SEQ ID NO:l) or a fragment or a variant thereof.

In some embodiments, the isolated protein comprises a C-terminal fragment of SEQ ID NO: 17 that comprises the amino acid sequence ADTRGILWAVE (SEQ

ID NO: 1 ) or a fragment or a variant thereof.

Another embodiment provides an isolated protein comprising the amino acid sequence ADTRGILWAVE (SEQ ID NO:l), or a fragment or variant thtreof, wherein the isolated protein further comprises an amino acid sequence heterologous to the amino acid sequence set forth in SEQ ID NO: 17.

In this context, by "heterologous" is that the amino acid sequence is not an amino acid sequence present in SEQ ID NO: 17. Suitably, the heterologous amino acid sequence is not present in any contiguous sequence of at least 5, 10, 15, 20, 25 or 50 amino acids of SEQ ID NO: 17. Non-limiting examples of heterologous amino acid sequences may include spacer amino acids that separate each peptide amino acid sequence, fusion partner or epitope tag amino acids, although without limitation thereto.

A yet further embodiment provides an isolated protein comprising the amino acid sequence ADTRGILWAVE (SEQ ID NO:l), wherein the isolated protein further comprises an amino acid sequence set forth in any one of SEQ ID NOS:7-16 or a fragment or variant of any of these.

Preferably, the isolated protein further comprises an amino acid sequence set forth in any one of SEQ ID NO:7 (VFSSPPFSNKPP), SEQ ID NO: 9 (VDRKEVAPVHES) or SEQ ID NO: 10 (PVSFSGPTKGTIT), or a fragment of variant of any of these.

It will be appreciated that the aforementioned isolated protein may comprise a single, contiguous amino acid sequence according to any of the following:

(i) an amino acid sequence of SEQ ID NO:l and/or a fragment or variant thereof;

(ii) an amino acid sequence of SEQ ID NO:l and/or a fragment or variant thereof and one or more amino acid sequences according to one or more of SEQ ID NOS:7-16 or preferably SEQ ID NOS:7, 9 or 10; (iii) (i) or (ii) wherein one or more of the amino acid sequences is repeated;

(iv) (i), (ii) or (iii) further comprising additional amino acids of the amino sequence set forth in SEQ ID NO: 17 and/or of an amino acid sequence heterologous to SEQ ID NO: 17.

The isolated peptide, fragment, variant and/or the isolated protein may be labeled, chemically-derivatized, cross-linked or otherwise modified to include one or other moieties or molecules in addition to the amino acid sequence of the peptide, fragment, variant and/or the isolated protein.

Non-limiting examples of chemical derivatives include amidated derivatives, derivatives made by iodination, peroxidation, succinylation, cross-linking, amino acid side chain modification and by addition of chemical couplings and protecting groups during solid phase synthesis, although withould limitation thereto.

As is well understood in the art, the label may be selected from a group including an enzyme, a fluorophore, a chemiluminescent molecule, biotin, avidin, a radioisotope or other label such as an epitope tag or fusion partner.

Examples of suitable enzyme labels useful in the present invention include alkaline phosphatase (AP), horseradish peroxidase (HRP), luciferase, β- galactosidase, glucose oxidase, lysozyme, malate dehydrogenase and the like. The enzyme label may be used alone or in combination with a second enzyme in solution or with a suitable chromogenic or chemiluminescent substrate.

Examples of chromogens include diaminobanzidine (DAB), permanent red, 3-ethylbenzthiazoline sulfonic acid (ABTS), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), nitro blue tetrazolium (NBT), 3,3 ',5,5 '-tetramethyl benzidine (TMB) and 4- chloro-1 -naphthol (4-CN) , although without limitation thereto.

A non-limiting example of a chemiluminescent substrate is Luminol™, which is oxidized in the presence of horseradish peroxidase and hydrogen peroxide to form an excited state product (3-aminophthalate).

Radioisotope labels may include ¹²⁵I, ¹³ Ί, ⁵¹ Cr and "Tc, although without limitation thereto.

Fluorophores may be a coumarin, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), allophycocyanin (APC), Texas Red (TR), TAMRA, LC red, HEX, FA , TET, ROX, Cy dyes such as Cy3 or Cy5 or R- Phycoerythrin (RPE) or derivatives thereof, although without limitation thereto.

Non-limiting examples of additional amino acids include linkers (such as the amino acid sequence GSGS-linked to biotin) and fusion partners and epitope tags that include metal-binding moieties such as polyhistidine (e.g. HIS₆), c-myc, influenza virus haemagglutinin and FLAG tags, although without limitation thereto.

Also provided is an isolated protein or peptide comprising a plurality of said peptides and/or variants. The isolated protein may be labeled, chemically-derivatized or otherwise modified, as hereinbefore described. A non-limiting example is an isolated protein comprising repeated or conctamerized amino acid sequences of the peptide or variant.

Another aspect of the invention provides a method of determining whether a subject has been exposed to, or infected by, C. trachomatis, said method including the step of determining whether a biological sample obtained from a subject comprises an antibody that binds an isolated peptide comprising the amino acid sequence ADTRGILWAVE (SEQ ID NO:l), or a fragment or variant thereof, wherein if the biological sample comprises the antibody the subject has been exposed to, or infected by, C. trachomatis.

Yet another aspect of the invention provides a method of determining whether a subject has, or is predisposed to, one or more C. trachomatis sequelae and/or a chronic C. trachomatis infection, said method including the step of determining whether a biological sample obtained from a subject comprises an antibody that binds an isolated peptide comprising the amino acid sequence ADTRGILWAVE (SEQ ID NO:l), or a fragment or variant thereof, wherein if the biological sample comprises the antibody the subject has, or is predisposed to, the one or more C. trachomatis sequelae or a chronic C. trachomatis infection.

As used herein, the terms "subject" or "individual" or "patient" may be used interchangeably to refer to any mammal, particularly a human, to be subjected to testing or diagnosis according to the invention disclosed herein.

Suitably, the subject is a female human.

Typically, the biological sample is a serum sample.

Suitably, the methods of these aspects includes the step of forming a complex between the isolated peptide, or an isolated protein comprising one or more peptide sequences according to SEQ ID NO:l, or a fragment or variant thereof, if present in a biological sample, such as a serum sample.

It will therefore be appreciated that the isolated peptide may facilitate detection of serum antibodies in a woman that indicates that the woman has previously been infected with, or exposed to, C. trachomatis.

Further to this, the isolated peptide may facilitate detection of serum antibodies in a woman that indicates that the woman has, or is predisposed to, one or more C. trachomatis sequelae.

Suitably, the isolated peptide may facilitate detection of serum antibodies in a woman that distinguishes a woman that has, or is predisposed to, one or more C. trachomatis sequelae and/or a chronic C. trachomatis infection, from a woman acutely infected with or by C. trachomatis.

By ^predisposed^"1 and "predisposition" is meant that the subject has a relatively increased probability or likelihood of, or susceptibility to, suffering from, C. trachomatis sequelae, whether or not the subject displays sequelae symptoms. In the context of the present invention, this relatively increased probability or likelihood of, or susceptibility to, suffering from C. trachomatis sequelae is determined or predicted with reference to that associated with one or more subjects that do not have detectable serum antibodies to the peptide of the invention or infertile women with no history of C. trachomatis infection or tubal damage.

A preferred method, of this aspect includes the step of forming a complex between the one or more antibodies present in a biological sample and an isolated peptide that comprises the amino acid sequence of SEQ ID NO:l or a fragment or variant thereof.

In a preferred embodiment, formation of a complex between the isolated peptide, or an isolated protein comprising one or more peptide sequences according to SEQ ID NO:l, or a fragment or variant thereof, is performed and/or detected by an immunoassay.

Non-limiting examples of immunoassays include an Enzyme Linked Immunosorbent Assay (ELISA), a radioimmunoassay (RIA), a magnetic bead immunoassay and a lateral flow immunoassay, although without limitation thereto. A preferred immunoassay is an ELISA, inclusive of particular forms of ELISA including an indirect ELISA, a sandwich ELISA and a competetive ELISA, although without limitation thereto. A preferred ELISA-based method includes the step of detecting formation of a complex between the one or more antibodies present in a biological sample and an isolated peptide that comprises the amino acid sequence of SEQ ID NO:l or a fragment or variant thereof. Suitably, the one or more antibodies in the complex are detected by a secondary antibody, typically a species- specific antibody (e.g. an anti-human Ig antibody).

Suitably, the secondary antibody is labelled, as hereinbefore described. In one embodiment, the secondary antibody is labeled with an enzyme (e.g HRP), although without limitation thereto. According this embodient, the method includes the step of providing an enzyme substrate (e.g a HRP substrate such as 3,3,5,5,-Tetramethyl- benzadine (TMB), although without limitation thereto) to thereby facilitate detection of complexes formed between the between the isolated peptide, or an isolated protein comprising one or more peptide sequences according to SEQ ID NO:l or a fragment or variant thereof.

It will also be appreciated mat according to the aforementioned methods, one or a plurality of other peptides may be used in combination with the isolated peptide of SEQ ID NO:l or a fragment or variant thereof. Non-limiting examples of other peptides are provided in Table 1. In particular embodiments, the other peptides respectively comprise the amino acid sequences of SEQ ID NOS:7-16, or fragments thereof, or more preferably comprising an amino acid sequence set forth in any one of SEQ ID NO:7 (VFSSPPFSN PP), SEQ ID NO: 9 (VDRKEVAPVHES) or SEQ ID NO: 10 (P VSFSGPT GTIT), or a fragment of variant of any of these.

In the particular context of an ELISA assay, a combination of peptides may be in any one or more of the following forms:

(a) different, individual peptides, each in the same well;

(b) different, individual peptides, some of which are in the same well, others in one or more other wells;

(c) different, individual peptides, each in respective wells;

(d) a single, contiguous isolated protein as disclosed herein comprising a plurality of the same or different peptides; or

(e) a combination of one or more of any of (a)-(d). It will be appreciated that a combination of peptides may be particularly useful by increasing sensitivity and/or specificity. For example, non-reactivity or weak reactivity with a single peptide may be read as a false negative but for reactivity (even weakly) with one or more other peptides in combination. Furthermore, reactivity with a plurality of peptides (even weakly) reduces the probability of false positive results that might be associated with detecting only a single antibody. In this regard, a "total score" may be produced based on reactivity with multiple peptides that gives greater confidence that the sample is positive or negative for antibodies indicative of C. trachomatis infection.

A preferred ELISA assay is in a "multiwell" format comprising a plurality of different individual peptides, each in separate wells, essentially as set forth in (c) above.

A still yet further aspect of the invention provides a composition or kit for use in detecting antibodies that bind an isolated peptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment or variant thereof

Suitably, the composition or kit comprises the isolated peptide that comprises the amino acid sequence of SEQ ID NO:l or a fragment or variant thereof, or an isolated protein comprising one or a plurality of said amino acid sequences.

It will also be appreciated that the composition or kit may comprise one or a plurality of other peptides in combination with the isolated peptide of SEQ ID NO: 1 or a fragment ot variant thereof. Non-limiting examples of other peptides are provided in Table 1. In particular embodiments, the peptides respectively comprise the amino acid sequences of SEQ ID NO:7-16, or fragments or variants thereof, or more preferably one or more peptides comprising an amino acid seqeunce set forth in any one of SEQ ID NO:7 (VFSSPPFSNKPP), SEQ ID NO: 9 (VDRKEVAPVHES) or SEQ ID NO: 10 (PVSFSGPTKGTIT), or a fragment of variant of any of these.

Typically, the composition or kit comprises one or more detection reagents that facilitate detection of an antibody-peptide complex formed between the isolated peptide or protein and one or more antibodies present in a biological sample. Detection regents may include a secondary antibody which is capable of binding the one or more antibodies present in a biological sample (which itself may be labelled, such as with an enzyme) and, optionally, one or more other reagents such as enzyme substrates, buffers, diluents, detergents and/or blocking agents, although without limitation thereto. In one embodiment, the composition or kit is suitable for use in an ELISA assay. Typically, the ELISA assay detects binding between one or more antibodies present in a biological sample and an isolated peptide that comprises the amino acid sequence of SEQ ID NO:l or a fragment or variant thereof and, optionally one or more other peptides such as set forth in SEQ ID NOS:7-16. For example, the one or more other peptides may include SEQ ID NO:7 (VFSSPPFSNKPP), SEQ ID NO: 9 (VDRKEVAPVHES) or SEQ ID NO: 10 (PVSFSGPTKGTIT), or a fragment of variant of any of these.

It will be understood by persons skilled in the art that ELISA assays may be performed in any of a number of formats.

A preferred ELISA assay detects binding between the one or more antibodies present in a biological sample and the isolated peptide that comprises the amino acid sequence of SEQ ID NO:l or a fragment or variant thereof, optionally including one or more other peptides set forth in SEQ ID NOS: 7-16, such as SEQ ID NO:7 (VFSSPPFSNKPP), SEQ ID NO: 9 (VDRKEVAPVHES) or SEQ ID NO: 10 (PVSFSGPTKGTIT), or a fragment of variant of any of these, wherein the one or more antibodies are detected by a secondary antibody. Suitably, according to this embodiment the composition or kit further comprises an enzyme-labeled secondary antibody (e.g a HRP-labeled anti-human antibody) and an enzyme substrate (e.g a HRP substrate such as 3,3,5,5,-Tetramethyl-benzadine (TMB), although without limitation thereto.

By way of example only, alternative ELISA assays could be a "sandwich" ELISA or a "competitive" ELISA, as are well understood in the art.

Optionally, the composition or kit may further comprise one or more reaction vessels such as one or more multiwell plates.

As previously described, a preferred ELISA assay is in a "multiwell" format comprising a plurality of different individual peptides, each in separate wells, essentially as set forth in (c) above.

Yet another aspect of the invention provides a monoclonal antibody that binds, recognizes and/or is raised against an isolated peptide comprising the amino acid sequence of SEQ ID NO: 1 , or a fragment or variant thereof.

In this context, a monoclonal antibody includes monoclonal antibody fragments and includes recombinant antibodies and fragments thereof, Fab and Fab'2 fragments, diabodies and single chain antibody fragments (e.g. scVs), although without limitation thereto.

In some embodiments, the monoclonal antibody may comprise a label selected from a group including an enzyme, a fluorophore, a chemiluminescent molecule, biotin, radioisotope or other label such as an epitope tag or fusion partner, as hereinbefore described.

A further aspect of the invention provides an isolated nucleic acid comprising a nucleotide sequence encoding the isolated peptide of SEQ ID NO:l or a variant or fragment thereof as disclosed herein, or encoding the isolated protein comprising one or a plurality of said peptides, fragments and/or variants disclosed herein

The term "nucleic acid" as used herein designates single-or double-stranded mRNA, RNA, cRNA, RNAi, siRNA and DNA inclusive of cDNA, mitochondrial DNA (mtDNA) and genomic DNA.

The invention also provides "genetic constructs" that comprise one or more isolated nucleic acids or fragments thereof as disclosed herein operably linked to one or more additional nucleotide sequences.

As generally used herein, a * 'genetic construct" is an artificially created nucleic acid that incorporates, and/or facilitates use of, an isolated nucleic acid disclosed herein.

In particular embodiments, such constructs may be useful for recombinant manipulation, propagation, amplification, homologous recombination and/or expression of said isolated nucleic acid.

As used herein, a genetic construct used for recombinant protein expression is referred to as an "expression construct", wherein the isolated nucleic acid to be expressed is operably linked or operably connected to one or more additional nucleotide sequences in an expression vector.

An "expression vector" may be either a self-replicating extra-chromosomal vector such as a plasmid, or a vector that integrates into a host genome.

In this context, the one or more additional nucleotide sequences are regulatory nucleotide sequences.

By "operably linked'^'' or "operably connected" is meant that said regulatory nucleotide sequence(s) is/are positioned relative to the nucleic acid to be expressed to initiate, regulate or otherwise control expression of the nucleic acid. Regulatory nucleotide sequences 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.

One or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, splice donor/acceptor sequences and enhancer or activator sequences.

Constitutive and inducible promoters as known in the art may be used. Constitutive promoters include, for example, CMV and SV40 promoters, although without limitation thereto. Inducible promoters may include, for example, nisin- inducible, tetracycline-repressible, IPTG-inducible, alcohol-inducible, acid-inducible and/or metal-inducible promoters, although without limitation thereto.

In one embodiment, the expression vector comprises a selectable marker gene. Selectable markers are useful whether for the purposes of selection of transformed bacteria (such as bla, kanR, ermB and tetR) or transformed mammalian cells (such as hygromycin, G418 and puromycin resistance).

Suitable host cells for expression may be prokaryotic or eukaryotic, such as bacterial cells inclusive of Escherichia coli (DH5a for example , yeast cells such as S. cerivisiae or Pichia pastoris, insect cells such as SF9 cells utilized with a baculovirus expression system, or any of various mammalian or other animal host cells such as CHO, BHK or 293 cells, although without limitation thereto.

So that the invention may be readily understood and put into practical effects, reference is made to the following non-limiting examples. EXAMPLES

EXAMPLE 1

MATERIALS & METHODS

Bioinformatics analysis to predict peptides with C. trachomatis specificity and putative B cell antigenicity

C. trachomatis proteins were selected for this study which had previously been identified as highly immunoreactive, when screened against serological positive females with C. trachomatis infection or C. trachomatis-re\ated tubal factor infertility in previous investigations. These proteins were: HtrA, cHSP60, Ct443, and ^Q381 [9-12]. A protein unique to Chlamydia pneumoniae (Cpn0236) was also included in the study as a negative control [13].

The protein sequences were analysed using a series of in silico bioinformatic analyses to identify potential linear B cell epitopes, and the specificity of these epitopes to C. trachomatis. The sequences of the proteins of interest were screened for predicted linear B cell epitopes using BepiPred algorithm software [14], confirmed with antigenicity prediction software [15], and hydrophilic domains identified to avoid transmembrane domains [16].

Candidate C. trachomatis HtrA, cHSP60, CT443, and CT381 linear B cell epitope peptides were screened for sequence specificity against the NR database, specifically against C. pneumoniae by BLAST and were selected if E values < 0.0004 for C. trachomatis and > 0.1 for C. pneumoniae were reached. Regions that did not show specificity to C. trachomatis were excluded from this study. The predicted epitopes were then used to design a series of peptides (as outlined in Table 1) which include partial or complete sequences of these epitopes. In some cases the epitopes for HtrA and HSP60, two of the most highly reported immunogenic antigens for Chlamydia, were used to test partial epitope peptides and also to 'walk' through the larger predicted epitope regions. These include the peptides 6-10, 15-18, 23-28, 32-34, and 37-43.

Analysis of Epitope Array to identify epitopes and development of ELISA

The peptide epitopes were commercially synthesized using solid phase synthesis onto a Biotin-SGSG motif (Mimotopes, Melbourne, Australia). All peptides were solubilized in 50% isopropanol overnight on a gentle rocker at room temperature. The initial screening ELISAs were conducted using the complete peptide epitope array against 39 participants across the cohorts.

The ELISAs were conducted using streptavidin coated plates (Reacti-Bind Streptavidin High Binding Capacity Coated 96-well Plates, ThermoScientific, Australia), and the SuperBlock Blocking Buffer in PBS (Pierce, Australia). The peptides were coated onto the plates at approximately 0.15 μg per well for one hour at room temperature in PBS/0.1% Tween 20, the plates were washed four times in PBS 0.1% Tween 20, and then blocked overnight in SuperBlock buffer with 0.1% Tween 20 at 4°C. The participant sera was added to the wells at dilutions of 1/200 and 1/1000 (in Superblock Buffer PBS with 0.1% Tween 20) and incubated for one hour. The plates were washed five, times in 2 x PBS with 0.1% Tween 20. The secondary antibody used was goat anti-human IgG-HRP (Invitrogen, Australia) at a dilution of 1/15000 in SuperBlock Blocking Buffer PBS with 0.1% Tween 20. The plates were developed by the addition of 100 μg/ml of 3,3,5,5,-Tetramethyl- benzadine (TMB) (Sigma Aldrich, Australia) in DMSO dissolved in phosphate citrate buffer with sodium perborate (Sigma Aldrich, Australia), incubated for 10 mins at room temperature and stopped by the addition of 1.0M H2SO4.

ELISA plates were read on Bio-Rad xMark Microplate Spectrophotometer at 450 nm. These ELISAs were conducted by screening the whole array against each participant sera with primary and secondary antibody only controls on each plate. The peptides which fit the criteria of being potentially able to be further developed in the initial analysis were then selected for further analysis. Optimized assays were attempted including further serological dilution series and a variety of alteration in washing conditions. The peptide 11 (SEQ ID NO:l) ELISA was conducted as per the above protocol except that 0.2 μg of peptide was coated to each well.

Participant Cohort collection and definitions

Participants (female) were recruited as a part of the Queensland Chlamydia Research Network which includes all Queensland Health Sexual Health Clinics, QUT Health Services Medical Centre, The Wesley Reproductive Medicine and Gynecological surgery unit. The participants were fully informed and consented prior to participation in the study. Ethical approval for the study was obtained via the following Human Research Ethics Committees QUT Human Research Ethics Committee approval number 080000268, Prince Charles Hospital Human Research Ethics Committee approval number EC2809, Ipswich and West Moreton Health Services District Human Research Ethics committee approval number (10-09), Gold Coast Hospital District Human Research Ethics Committee approval number (200893), Cairns Sexual Health Clinic (HREC/09/QCH/4-554), and The Wesley Hospital Human Research Ethics Committee (2008/02).

The following data was collected from all participants: age, number of sexual partners, fertility status (if known), self reported and patient chart reported history of sexually transmitted infections, current and previous C. trachomatis infections, contraceptive use and type. Participants sourced from the Wesley Reproductive Medicine and Gynecological surgery unit were also asked to allow their gynecologist to provide additional data relating to their infertility status and infertility cause. Those patients with tubal factor infertility and positive serology to C. trachomatis using the below tests were considered to belong in the sequelae cohort. Participants from this clinic were all tested for C. trachomatis infection history using both the MEDAC and Bioclone C. trachomatis ELISAs (sourced from Bioclone, Sydney, Australia and MEDAC sourced from Biocene, Sydney Australia). Participants recruited at this clinic who were seropositive to C. trachomatis but were recorded to have infertility for a reason other than tubal factor were not used in this study due to the potential for their infertility to still relate to C. trachomatis immunopathology. Acute participants were those who had a history of single treated C. trachomatis infection (PCR diagnosis) with no reported sequelae and multiple infections were those who had a history of more than one treated C. trachomatis infection (PCR diagnosis) with no reported sequelae. These two cohorts were generally recruited at the Sexual Health Clinics. The C. trachomatis sequelae cohort included women who had a history of PID, ectopic pregnancy or tubal factor infertility with a history of C. trachomatis infection (either by participant chart recorded PCR positive previous infections or C. trachomatis positive serology by MEDAC and Bioclone). The negative control cohort included women attending the IVF clinic who were infertile but did not have tubal factor infertility; these participants were all negative for C trachomatis serology by the MEDAC and Bioclone tests.

Participants (female) were also recruited from patients attending the Gynecology outpatient department at the Sarfdarjung Hospital, New Delhi, India. Participants were informed and consented to participate in the study. The study was approved by the Sarfdarjung Hospital Human Research Ethics Committee. The participant data collected included; age, regularity of menstrual cycle, years of infertility (if infertile), clinical details such as cervicitis, bacterial vaginosis, Mycoplasma sp. culture, Ureaplasma sp. by culture, bacterial vaginosis, and C. trachomatis infection status by direct fluorescence assay and PCR from swabs.

Participants from this study were grouped into cohorts depending on if they had primary or secondary infertility, or were attending the clinic for cervicitis. Each of these cohorts were analyzed also according to if they had a current detected C. trachomatis genital infection. RESULTS

Screening new peptide antigens using ELISA confirms bioinformatic predictions and identifies potential new antigens for Chlamydia antibody testing

The peptide array (55 unique peptides) was developed using a series of bioinformatic analyses of the most commonly reported proteins to have a serological response in C. trachomatis participants. Proteins previously identified as positive during screening or proteomic studies to be frequently identified when screening with participant sera were selected (HtrA, Hsp60, CT443, and CT381), along with a C. pneumoniae specific protein as a negative control (CP0236). MOMP was not included for this study given the existing peptide ELISAs based on this protein and the serovar specific sequence variability in this protein. These proteins were then searched bioinformatically for B cell epitopes, as outlined in the Materials and Methods. Predicted epitopes were then searched by BLAST to reduce the pool of potential epitopes to only those which had high sequence specificity to C. trachomatis. An initial screening assay was conducted to identify peptides which may be useful detectors of the sequelae disease cohort.

The peptide array was screened in duplicate against 39 participant sera belonging to the five cohorts; sequelae (tubal factor infertility, ectopic pregnancy, PID), acute, multiple, negative (infertile but C. pneumoniae seropositive), and negative (both C. trachomatis and C. pneumoniae seronegative, infertile but no tubal damage). The raw values for each epitope from each participant in each cohort were then analyzed (Table 1). The data was analyzed by ANOVA, comparing the data for each cohort to either sequelae or negative groups.

Only the peptide HSP60-E2 (SEQ ID NO: 7) showed a significant difference

(p<0.05, sequelae c.f. acute) when the results were tested for significant differences between the cohorts during this initial screening ELISA. However, these ELISAs were analysed with a set of triage rules were applied to select peptides to screen further (detailed below).

This initial screen does not contain enough samples to allow robust statistical analysis of the performance of the epitope prediction software, however trends consistent with the predictions were observed. We examined the results for only those peptides which had a BLAST E score against the C. trachomatis genome of less than 0.0008 (i.e. likely highly specific to C. trachomatis). From these 13 epitopes, 6 were positive for the initial triage criteria (explained further below) for the potential to differentiate between sequelae and negative cohorts; 5 of these 6 contained predicted epitope sequences for the entire length of the peptide. On the converse, the 6 peptides which had a BLAST score greater than 0.01 against the C. trachomatis genome, only one was considered positive in the initial triage to compare responses between sequelae and negative cohorts (Table 4).

In order to identify which peptides have the potential for further assay optimization for specific detection of the sequelae cohort a set of criteria were used to select the peptides to develop further. The three criteria were: 1. achieve a higher average absorbance in the sequelae cohort compared to the negative cohorts (a difference off of >0.015 was chosen), 2. to avoid false positive detection due to prior C. pnuemoniae infection (<0.015 difference between the two negative cohorts), and 3. distinguish sequelae from acute participants (>0.015 different in absorbance between the two cohorts) (Table 4). These three criteria identified the

following peptides; 11 (HtrA; SEQ ID NO:l), 48 (HSP60; SEQ ID NO:8), 51 (Ct443; SEQ ID NO: 11), 49 (Ct443-a3; SEQ ID NO:9) and 52 (Ct381; SEQ ID NO: 12). Peptide 11 comprises an amino acid sequence set forth in SEQ ID NO. l. Peptide 11 (SEQ ID NO: 1) putatively comprises the epitope sequence ADTRGILVV (SEQ ID NO:2). Peptide 47 in Table 1 (SEQ ID NO: 10) contains the predicted epitope VFSSPP (SEQ ID NO:13).Peptide 48 comprises the amino acid sequence SLSSSPEHSQEE (SEQ ID NO:8) comprising the putative epitope SLSSSPE (SEQ ID NO: 14). Peptide 51 comprises the amino acid sequence LTVPVSDTENT(SEQ ID NO: 15). Peptide 52 putatively comprises the epitope sequence VGIGVASDRPAL (SEQ ID NO: 16).

Peptide 11 ELISA is an effective serological diagnostic for chlamydial tubal factor infertility

In order to optimize the serological assay, performance of the different assay conditions trialed with the different sera dilutions for each of the four peptides were assessed by area under the curve analysis. Specificity was controlled at >95% to establish absorbance (450nm) thresholds, as this study aimed to develop a diagnostic to detect those women who have sequelae from C. trachomatis infection.

Peptide 11 (SEQ ID NO:l) comprising the putative epitope sequence of SEQ ID NO:2, showed the most rigorous performance and hence only the data from this peptide is shown here. Figure 1 shows the receiver operator characteristic curve for the sera dilution of 1/200 comparing participants from sequelae and negative infection cohorts. The dilution series did not significantly change the accuracy of the test as there was negligible difference in area under the curve. The major difference between the dilutions was one of practicability, as the absorbance threshold was to be 0.184 at a dilution of 800 and 0.296 at a dilution of 200 which achieves a better signal to noise threshold for assay development.

The absorbance threshold for a dilution of 200 was then used to compute the odds ratios of peptide 11 with other participant cohorts. As shown in Table 2, the peptide 11 ELISA is able to be used to discriminate participants in the sequelae disease cohort compared to negative control cohorts with an odds ratio of 16.3 (95% confidence interval 1.63-160). The bioinformatic prediction that the test will not be influenced by C. pneumoniae serological status is also supported by its performance against the negative cohort which includes 7 C. pneumoniae seropositive participants.

The test tended to produce positive odds ratios for multiple or acute infections compared to the negative cohort, although the 95% confidence interval included no change. Comparison of the multiple infection and the negative cohorts had an odds ratio of 4.07 (95% confidence interval 0.38-43.4).

The test parameters were also analyzed separately for a potential application in a general practice setting with the potential to distinguish between sequelae and acute infection cohorts. A 95% specificity cutoff for this comparison yielded an absorbance threshold of 0.408 (Table 2). This absorbance threshold had an odds ratio of 8.10 (95% confidence interval 0.75-87.3) for detecting sequalae over the acute infection cohort.

Testing the peptide 11 assay as a serological diagnostic using cohort of women attending a gynecology clinic in India

The Peptide 11 ELISA was then tested against participants recruited by the Sarfdarjung Hospital Gynaecology Clinic, South Delhi. The peptide 11 ELISA was tested using 129 women attending this clinic using the 200 fold sera dilution with the designed 95% specificity absorbance threshold of 0.296 to determine those women positive for sequelae. A brief gynecological history was recorded (as outlined in Materials and Methods) and the women were screened for current genital infection status. The women who tested positive in the peptide 11 ELISA compared to those who tested negative were analyzed for any potential confounders which may have influenced their infertility status or the test performance. There was no statistical significance in age of women who tested positive and those who tested negative (p=0.3458, 28.43 average years for peptide 11 positive c.f. 30.16 average years for peptide 11 negative), or in the patient reported years of infertility (p=0.6802, 6.47 compared to 7.11 years for peptide 11 positive group).

Furthermore there were no significant differences in those women who were positive in the peptide 11 ELISA and whether or not they had current genital infections (Mycoplasma, or Ureaplasma). Interestingly, women who tested positive in the peptide 11 ELISA had an odds ratio of 4.19 for also having bacterial vaginosis (95% confidence intervals 0.5-156.29). In spite of there being no difference in the age of women who tested positive compared to those who tested negative; all of the women who were peptide 11 positive were women with primary infertility (Table 3). Those women who were positive in the peptide 11 ELISA had an odds ratio of 8.8 (95% confidence interval 0.5-156.29) of having primary infertility compared to all other women in the study. Furthermore, women who tested positive to the peptide 11 ELISA demonstrated a significantly higher proportion of those with primary infertility and C. trachomatis infection or primary infertility and cervicitis (Table 3; P value= 0.039, Fisher's exact test). Whilst a large number of the women recruited at this clinic were positive for a current C. trachomatis infection (47 positive by both urine DFA and PCR), 2 of the 9 women who tested positive using the peptide 11 test also had a current infection further supporting that the design of the test does not bias towards detection women with acute infections. MEDAC MOMP ELISA detected 5 infertile positive women (absorbance above 1.1) including; 2 women with C. trachomatis and cervicitis, 2 women with primary infertility, and 1 primary infertility with C. trachomatis infection, who was also detected by the peptide 11 assay. Sensitivity and specificity of peptide 11 or MEDAC performance relative to the gold standard of laparoscopy or hysterosalpingogram was not possible on these patients.

The data in FIGS 2 and 3 also show that Peptide 11 (SEQ ID NO:l) in combination with peptides cHsp60-e2 (PVSFSGPTKGTIT; SEQ ID NO:7) and 443- n2 (VFSSPPFSN PP; SEQ ID NO: 10) showed a significant ability to distinguish the difference between acute infection and sequelae. Furthermore, these peptide combinations were used to evaluate the difference in the IgG subclasses (IgGi, IgG2, IgG₃ & IgG₄) between acute infection and sequelae. IgG₃ was found to be more predominant as compared to other subclasses. No significant difference was observed between IgG subclasses of patients with acute infection and sequelae. DISCUSSION

This study shows for the first time that a peptide from C. trachomatis HtrA could be developed into a specific diagnostic to detect serious sequelae from this infection. Women who reacted positively in the peptide 11 ELISA developed during this study had a 16.3 odds ration of having C. trachomatis sequelae (tubal infertility, ectopic pregnancy, or PID) (specificity 95% and sensitivity 46%). The test also showed the potential to distinguish these women with sequelae from women with a history of C. trachomatis single treated infection or multiple infections,

although, the cohort numbers were not large enough in this study to provide statistical validity.

This is an important finding as very few studies reporting new antigens for diagnosis of tubal factor infertility report the performance of the test against infection cohorts. Not only do these data support the use of the peptide 11 ELISA for diagnosis of tubal factor infertility during the IVF work up, the data also imply that it may be possible to further develop this assay and screen larger numbers to successfully distinguish between the infected or history of infection cohorts. This aspect of the test will be developed further with a view to making this assay useful for both epidemiological and clinical applications.

The strategy of using a combination of previous proteomic data on immunogenic antigens and bioinformatic tools to select epitopes for peptide ELISA data was supported by the findings in this study. These findings further validate the attempts within the field to search for new candidate antigens to develop a new Chlamydia antibody test to detect those women with Chlamydia related tubal infertility.

The peptide 11 (SEQ ID NO:l) ELISA was tested in another setting and it was found that 9 out of 129 women attending a Gynecology clinic in India would be predicted to have tubal infertility using this assay. Interestingly, all of these women had primary infertility. There were 47 women with a current C. trachomatis infection of the 129 women recruited for this study (36.4%). This prevalence is slightly higher than a previously reported prevalence of C. trachomatis positive women at another clinic in Delhi, however there is potential for a recruitment bias of some kind in our study as the clinicians were aware that this is a Chlamydia research study (24-30%) (PCR diagnosis) [17]. C. trachomatis infection prevalence in populations in India (generally sexual health clinic, gynecology clinic or sex worker screening studies) varies from 7%-30% [18-19]. It is more difficult to estimate the likely percent of infertility that relates to CA/amj¾//a-mediated tubal factor infertility. However, if we use the data from two different prospective studies where women attending IVF clinics were recruited, calculate the percent of participants with tubal infertility and a positive antibody testing for a history of Chlamydia infection and assume this was the cause of the tubal infertility, we can predict the overall percent of women attending IVF clinics due to Chlamydia related tubal infertility. In a study based in Denmark this calculation would predict that 6.9% of infertile women attending the IVF clinic were due to C. trachomatis tubal infertility [20]. For a study in China the calculation predicts that 7.6% of women attending the IVF clinic had C. trachomatis tubal infertility [21]. The participants attending the clinic in India for this current investigation do not have access to laparoscopy or hysterosalpingography

technologies, meaning it is unfortunately not possible to validate the results of the peptide 11 assay in this second cohort. However, 7.0% of the participants attending the clinic, all with primary infertility, were positive by this assay.

EXAMPLE 2

MATERIALS AND METHODS

Study population

The study population consisted of 354 women recruited from the following health and IVF clinics. Participants are recruited from several Australian fertility clinics which include Wesley IVF (Brisbane), Monash IVF (Melbourne) Prince Charles Hospital, Ipswich and West Moreton Health Services District, Gold Coast Hospital District, Cairns Sexual Health Clinic and The Wesley Hospital. In addition to characterizing the patients based on disease state, other factors such as age of the patient, number of sexual partners, previous C. trachomatis infections, previous sexually transmitted infections, smoking habits and previous record of infertility such as ectopic pregnancy and tubal factor infertility will be considered while developing the assay. This would enable the identification of confounding factors that may affect the outcome of the assay.

Infertile cohort (Cohort 1)

Infertility in patients is defined as the inability of the patient to get pregnant after a period of 1 year. Patients belonging to infertile cohort were recruited from Monash IVF (Melbourne) clinic and Wesley IVF. A total of 100 patients will be recruited. With the consent of patients attending the clinic, the blood sample was collected from 26 patients (ongoing recruitment). The serum was later separated from the blood and shipped to the lab and fertility clinic chart auditing was conducted to identify their tubal fertility status was conducted with their consent. The patients were asked to complete a patient history that entails their fertility and gynaecological details. Through this patient history, information regarding previous C. trachomatis infection, behavioural variables, age and ethnicity were obtained. The patients were separated into cohort D and cohort E based on their aetiology. The patients were diagnosed with laproscopy, hysterosalpinography and hysteroscopy. Cohort D comprised of those patients with patent tubes (n=4) and cohort E comprised of patients without patent tubes (n=26). In addition to tubal patency, some of the patients in cohort D were also diagnosed with polycystic ovary syndrome and fibroids. All four patients had undergone previous tubal patency investigation. Out of 4 patients, 3 patients have previously been diagnosed with partially patent tubes and bilateral tubal occlusion. Cohort E comprises of women with infertility caused due to endometriosis, idiopathic PCO (polycystic ovary syndrome), fibroids or due to unknown reasons. Their patient history include the gynaecological surgeries they have undergone previously; such as endometrial polyp, fibrioids, endomteriosis, ectopic pregnancy, miscarriages, polycystic ovaries and patent tubes.

Acute and negative cohort (Cohort 2)

The patients with acute C.trachomatis infections were recruited from health clinics in Queensland. Patients with acute infections are PCR diagnosed C.trachomatis genital infections with minor or no symptoms. The clinics include Prince Charles Hospital (Human Research Ethics Committee approval number EC2809), Ipswich and West Moreton Health Services District (Human Research Ethics committee approval number (10-09)), Gold Coast Hospital District (Human Research Ethics Committee approval number (200893)), Cairns Sexual Health Clinic (HREC/09/QCH/4-554), Monash IVF, Clayton and Hawthorn and The Wesley Hospital Human Research Ethics Committee (2008/02). Ethical approval for the study was obtained via Queensland University of Technology Human Research Ethics (080000268). Through PCR-diagnosis, the sera of those patients positive for C.trachomatis infection were collected. Additionally, they were required to complete a patient history form which provided information regarding their fertility issues and history of chlamydial infection. The negative cohort which includes fertile patients without C.trachomatis infection has also been recruited from the aforementioned clinics. The sera were collected from women between the ages of 26-40 years who have undergone current spontaneous pregnancy and were able to get pregnant within less than a year of trying. The patient histories with their gynaecological histories were also recorded. International test cohort (Cohort 3)

249 serum samples were collected from patients attending clinics in Samoa. QUT is blinded until the serological analysis is completed.

Serological analysis of cohorts

The serum samples from cohort 1, 2 and 3 were tested for C.trachomatis serology using commercial serological kit and In-house assay, both of which use an

ELISA platform. The performance of peptide combination assay and commercial serological kits were compared against cohort 1 , which comprises of TFI patients.

Commercial serological kits were used to further support the patient history and PCR diagnosis results. Following estimation of diagnostic accuracy of peptide 11 and the peptide combination assays, the assay is run on test cohorts, 2 and 3 to validate its diagnostic performance.

Commercial serological kits

C. trachomatis infections in each cohort were determined serologically using commercial kits. Chlamydia trachomatis IgG-p-ELISA Medac that uses a synthetic peptide from the immunodominant region of MOMP was used to determine acute infection in patient cohort. The optical density of the test plates were read in a spectrophotometer and based on the cut-off value, the samples were categorized as positive and negative. Samples positive for chronic C. trachomatis infection were determined using the cHSP60-IgG-ELISA medac. It utilises recombinant heat shock protein 60 from C. trachomatis as antigen and it detects IgG antibodies to cHSP60 from C. trachomatis. The kit predominantly detects antibodies to C. trachomatis and it is instrumental in detecting C. trachomatis - induced tubal damage. The patient cohorts were also tested for the presence of C.pneumoniae using the Chlamydia pneumoniae-lgG-ELlSA plus medac kit. The samples that gave an absorbance reading greater than 1.1 on Chlamydia trachomatis IgG-p-ELISA Medac were considered positive for TFI [7].

In-house serological assay

In addition to commercial assay, the cohorts were further defined using an in- house ELISA based on peptide 11 (SEQ ID NO:l), a peptide derivative of immunodominant antigen HtrA. Peptide 11, which was designed in our lab, was selected from series of 55 unique peptides that showed a serological response to C. trachomatis [22]. Peptide 11 was derived from HtrA using a combination of previous proteomic data on immunogenic antigens and bioinformatic tools to select for peptide epitopes [23]. The peptide has been reported to identify patients with C.trachomatis-related infertility. The specificity was controlled at >95% with the absorbance threshold of 0.408 for C. trachomaUs-related infertility cohorts [22], Peptide 11 has a sensitivity of 46% and a specificity of 95%.The peptides epitopes were synthesized commercially using solid phase synthesis onto a Biotin-SGSG motif (Mimotopes, Melbourne, Australia). The peptide was solubilized in 50% isopropanol overnight to make up a concentration of Img/mL. The sequence of peptide 11 comprising the C-terminal SGSG motif is SGSGADTRGILVVAVE-NH2 (SEQ ID NO:67).

ELISA based on peptide 11 in combination with other immunogenic peptides

In order to improve the sensitivity of the assay, a peptide based ELISA was designed with peptide 11 in combination with peptides from other immunogenic antigens such as Hsp60 and CT 443. After preliminary analysis, the peptides hsp60- e2 (SEQ ID NO:7) and 443-n2 (SEQ ID NO: 10) were selected based on their ability to effectively differentiate acute infections from sequelae. The combinations include peptide 11 /hsp60-e2 andpeptide 1 l/hsp60-e2 /443-n2. The biotinylated peptides were coated on streptavidin plates. Peptide 11 (lmg/mL) was diluted in lx PBST (1:200), peptidel l

(lmg/nlL)[0.25μg/well for each peptides] combination and peptide H(lmg/mL) hsp60-e2(lmg mL)/443-n2 (lmg mL)

for each peptide] combination were diluted in IXPBST at 1 :100 dilution. The peptides were incubated for 1 hour at room temperature. The sera was diluted in superblock blocking buffer PBS with 0.1% Tween20 at 1:200 dilutions, and incubated for 1 hour at 37°C. The secondary antibody used was goat anti-human IgG HRP at a dilution of 1/15,000 in superblock blocking buffer PBS with 0.1% Tween 20. The plate was developed by addition of 100μg/mL of 3,3,5,5-Tetramethyl benzadine (TMB) in DMSO dissolved in phosphate citrate buffer with sodium perborate and incubated at 10 minutes at room temperature. The reaction was stopped with 1M H₂SC>4 and the plates were read on Bio-Rad xMark microplate spectrophotometer at 450nm. During optimisation the influence of several parameters were considered, such as peptide concentrations and combinations, dilution of secondary antibodies and peptide solubility.

Validation of ELISA for detecting patients with Ctrachomatis-related infertility

The area under the curve (AUC) and the corresponding cut-off value for peptidel l, peptide 1 l/hsp60-e2 and peptide 1 l/hsp60-e2/443-n2 ELISA were determined by running the assay on TFI patients alone. The assay would be run on cohort 2 and cohort 3 and the specificity and sensitivity would be determined by applying the cut off value obtained through cohort 1. The assay would further be validated by measuring the inter- and intra-assay variability. 6 peptide combination assays were conducted with 9 replicates and the standard error difference obtained between the assays for the samples would determine the degree of inter and intra- assay variability.

Multiwell assay

The samples were assayed against peptidel l, hsp60-e2, 443-n2, peptide

1 l hsp60-e2 and peptide 1 l/hsp60-e2/443-n2 to compare the ability of individual peptides to identify patients with Ctrachomatis-related infertility with peptide combinations. Each well was coated with individual peptides (Peptidel l, hsp60-e2, 443-n2) or combination of peptides (peptide 1 l/hsp60-e2/443-n2, peptide 1 l/hsp60- e2) and tested against 6 patients from each cohort with TFI, acute C.trachomatis infection and negative control (healthy control, TFI patients without C.trachomatis infection and fertile patients). The multi-well assay was conducted to determine the compare diagnostic performance of individual peptides and peptide combinations in detecting patients with Ctrachomatis-related TFI. The ELISA conditions as same as outlined above.

Statistical analysis

All statistics (relative risk, sensitivity, specificity, chi squared statistics and forest plots) were calculated in the R statistical environment (3.0.1) using the 'EpiR' (0.9-48) and 'metafor' package (1.9-1) for conducting meta-analyses in R (Viechtbauer 2010). Mixed-effects models were used to combine cohort data using a fixed-effect model with a restricted maximum likelihood estimate of the log odds ratio [24]

The inter and intra assay variability was determined as follows, linear-mixed effects models were calculated for the different assays taking into account fixed differences in sample absorbance with plate-to-plate differences as a separate stochastic variable. The resulting inter and intra assay standard errors were calculated as restricted maximum likelihood estimates. All calculations were performed in the R statistical environment (v 3.0.1) using the Linear and Nonlinear Mixed Effects Models package 'nlme* (v 3.1 - 111 ) [25] .

RESULTS

Cohort characterization using commercial and in-house serological assay

Using the cut-off for peptide 11 established in Stansfield et al. [22], the sera from cohort 1 , 2 and 3 were assayed against peptide 11 and patients positive for Ctrachomatis-related TFI were identified. The combination of Medac MOMP/cHSP60 and the absorbance value of Medac MOMP >1.1 was used to assing the cohort for the Ctrachomatis-Telated TFI patients for those cohorts which do not have other data such as laparoscopy or hyesteroscopy. The results are shown in Table 5.

Small scale evaluation of peptide combination ELISA The small-scale evaluation of peptide combinations included determination of the specificity and sensitivity of peptide combinations against 6 patient samples from each cohort (Patients with C. trachomatis infections-related TFI, acute infections and no C. trachomatis infections). The peptide combinations included peptidel 1/443 -n2, peptidel l/443-a3, peptidel l/hsp60-e2, peptidel 1/443 -n2/hsp60-e2 and peptide 1 l/443-a3/hsp60-e2. Table 6 shows the area under the curve analysis and sensitivity and specificity for the aforementioned peptide combinations.

The accuracy of the assay is determined by the ROC values, which are greater than 0.5 for all peptide combinations; hence the tests, particularly peptide 11, peptidel l/443-n2/hsp60-e2, peptide I l/hsp60-e2 produce accurate results and give higher number of true positives. The specificity was 100% for all the peptide combinations and the sensitivity was highest for peptidel 1 (100%), followed by peptide 1 l/hsp60-e2 (83%) and peptidel 1/443 -n2/hsp60-e2 (67%). Based on these analysis, peptide 11, peptidel l/hsp60-e2 and peptide 1 l/hsp60-e2/443-n2 are identified for further assay development.

Large scale evaluation of peptide combination assays

Based on the results obtained for small-scale assay, the peptide ELISAs were applied to a large cohort of samples. A total of 354 samples were assayed against peptide 11, peptidel l/hsp60-e2 and peptidel l hsp60-e2/443-n2. The samples were recruited from Samoa (n=249), infertility clinic at Monash IVF (n=32), samples with acute C.trachomatis infections (n=50), fertile patients (n=28).

Diagnostic performance of commercial serological kit vs peptide 11 -based ELISA Diagnostic performance of peptide 11 combination ELISA

The diagnostic performance of peptide 11 combination ELISA were evaluated by determining the area under the curve, odds ratio and the specificity and sensitivity of detecting Ctrachomatis-related infertility in cohort 1, 2 and 3. Table 6 shows the area under the curve for peptide 11 and its peptide combinations. Those samples that were positive for MEDAC MOMP+cHSP60 were considered TFI positive; 58 patients from Samoa cohort, 2 from TFI Monash cohort and 1 from the fertile cohort were characterized as C.trachomatis sequelae. For the peptide 11 assay, patients were considered positive for TFI if the absorbance values were greater than 0.296 (Table 7). Patients whose absorbance values for Medac MOMP was greater than 1.1 were considered to be TFI positive (Table 8). The odds ratio and specificity and sensitivity of peptide 11 to detect TFI patients in each cohort were found to be quite superior to Medac assays (Table 7 and Table 8).

Diagnostic performance of Multi-well assay

Table 9 represents the performance all peptides and its combinations in identifying patients with C. trachomatis-rdated infertility (n=9 TFI positive; n=18 TFI negative). In comparison with peptide 11, peptide hsp60-e2 and peptide 443- n2showed higher sensitivity (89% and 1 respectively) and specificity (83% and 89% respectively) in distinguishing patients with C .trachomatis-related infertility from those that did not have the disease. By combining the peptides, an increase in specificity was observed for peptide 1 l/hsp60-e2. Although, an increase in specificity and sensitivity would be expected for triple combination, the peptide 1 l/hsp60- e2/443-n2 combination yielded lower specificity (78%) and sensitivity (83%). However, the combination has increased the sensitivity and specificity of the assay as compared to peptide 11. Additionally, the combinations have reduced the cut off- value, with pepl l/hsp60-e2 at 0.28 and peptidel l/hsp60-e2/443-n2 at 0.22.

Similar results have been obtained by Dubois et al.[26], wherein by mixing equal concentrations of overlapping 20mer peptides in the same ELIS A improved the sensitivity of double peptide combination, whereas in triple combination, the sensitivity appeared to have decreased. Combination of peptides in the same well increased specificity in several cases but it did not improve the sensitivity. The performance of peptide combination depends on the antigenicity of the epitopes.

Inter- and intra-assay variability

The diagnostic accuracy and performance of peptide combination assays were further evaluated by measuring the inter and intra-assay variability. From the inter and intra-assay standard error values (Table 10), it could be inferred that the intra assay variability was low (<5%). Although, there is a high variability in the absorbance values over 6 days, the standard error values are <10%, hence the assay could be considered reproducible and repeatable. As for the target cohort, patients with C.trachomatis-related infertility, the inter- variability assay. 9 samples from each cohort were assayed against peptide 11 and combination for 6 days. Further optimisation of peptide 11 to improve diagnostic performance and capability to scale up

Peptide solubility

Peptide 11 solubilisation is a time consuming process which may not be ultimately compatible with large scale production of the ELISA. Therefore peptide 11 alternatives and options for solubility were explored. Peptide 11 comprises a B cell epitope and is derived from HtrA of C. trachomatis. The peptide sequence is ADTRGILWAVE (SEQ ID NO:l). The peptide has been used in conjunction with other peptides derived from C.trachomatis immunogenic proteins, Hsp60 and Ct443. The sequence of peptide hsp60-e2 is VFSSPPFSNKPP (SEQ ID NO:7) and the peptide sequence of 443-n2 is PVSFSGPTKGTIT (SEQ ID NO: 10). The physical characteristics of N-terminally biotinylated and C-terminally amidated versions of these peptides are shown in Table 11.

Since the net charge of both peptide hsp60-e2 and 443-n2 are basic with 8% hydrophilic residues and 58% and 38% hydrophobic residues respectively, the peptides are soluble in water and weak organic acids such 20% acetic acid. If the peptide is not soluble , it could be soluble in 0.1% TFA/H₂0. As peptide hsp60-e2 contains 58% hydrophobic residues, it may be insoluble or partially soluble in aqueous solution and may need to be dissolved in stronger solvents such as DMF, acetonitrile, isopropyl alcohol, ethanol, acetic acid and DMSO. Peptide 11 has a net acidic charge and contains 25% hydrophilic residue and 58% hydrophobic residue. Since it is an acidic peptide, it should be readily soluble in aqueous solutions; however, due to higher proportion of hydrophobic residues, it might be completely soluble. This could be overcome by sonication or by addition of 0.1% NH₄OH drop wise.

Narita et al.[27] developed an equation that enables prediction of peptide solubility based on the frequency and the type of amino acid residues in a peptide. The equation involves the coil conformational parameter, Pc, which is defined as the frequency of each amino acid residue in the coil regions and the average frequency of all residues in coil regions. The <Pc> value determines the solubility of peptides in organic solvents. Peptide 11 has a <Pc> value of 0.8, peptide hsp60-e2 has a <Pc> value of 1.2 and peptide 443-n2 has a <Pc> value of 1.25. Peptides having a value below 0.9 of <Po (peptide 11) are much less soluble in solvents, whereas peptides with value greater than 0.9 are soluble in organic solvents (peptide hsp60-e2 and peptide 443-n2).

Thus peptide 11 might not be particularly soluble in aqueous or organic solvents, although it may be soluble in a basic solution such as 0.005M NaOH or 0.001M NaOH. Bioinformatic analysis also suggests the use of several variants of the peptide containing the peptide 11 epitope that could improve its solubility. Table 12 shows the peptide 11 variants (SEQ ID NOS: 5 and 6) and their chemical properties.

Overall, these studies have successfully developed a new peptide based ELISA which is highly specific for detection of women with C. trachomatis sequelae such as tubal factor infertility. In particular, the ELISA may best be practised in a multiwell format using the peptides of SEQ ID NOS:l, 7, 9 and 10 in respective, separate wells. The assay may be both a useful diagnostic for initial fertility work up, and may also be useful for implementation as a screening tool in epidemiological studies, or within general practice as an early warning system for women to plan their families.

Throughout the specification, the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Various changes and modifications may be made to the embodiments described and illustrated without departing from the present invention.

The disclosure of each patent and scientific document, computer program and algorithm referred to in this specification is incorporated by reference in its entirety.

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Table 1: Results of screening of peptide array against different participant cohorts

sequelae Neg Cpn pos multiple grouped Neg (n=H) (n=7) Neg (n=9) (n=6) acute (n=6) (n=16)

Peptide Sequence Protein

Peptide (epitope prediction in BLAST - BLAST -

ID bold) Ctrach Cpn av (sd) av (sd) av (sd) av (sd) av (sd) av (sd)

HtrA 0.172 0.176

1 SPMLGYSASK D 0.003 2.4 (0.059) 0.145 (0.079) (0.071) 0.163 (0.040) 0.169 (0.08) 0.163 (0.074)

0.157 0.151

2 ADICLAVSSGDQ 0.005 1.2 (0.057) 0.123 (0.049) (0.052) 0.152 (0.039) 0.169 (0.085) 0.139 (0.052)

0.171 0.165 0.175

3 AVSSGDQEVSQE 0.01 0.87 (0.065) 0.139 (0.069) (0.058) (0.0369) 0.183 (0.074) 0.154 (0.062)

0.169 0.167

4 EQQRPQQRDAVR 2.00E-04 1.7 (0.061) 0.140 (0.071) (0.059) 0.173 (0.047) 0.164 (0.067) 0.155 (0.064)

0.163 0.168

5 QKYTAKTVGLDP 0.002 0.006 (0.060) 0.126 (0.063) (0.060) 0.168 (0.039) 0.176 (0.087) 0.150 (0.063)

0.155 0.160

6 AISLMMPGTRVI 6.00E-04 0.12 (0.058) 0.126 (0.067) (0.062) 0.164 (0.032) 0.163 (0.079) 0.146 (0.064)

0.169 0.179

7 VTVTQIPTEDGV 0.003 0.036 (0.073) 0.134 (0.069) (0.072) 0.171 (0.033) 0.165 (0.065) 0.160 (0.073)

0.153 0.171

8 EDGVSALQKMGV 0.004 0.01 (0.054) 0.139 (0.050) (0.061) 0.181 (0.050) 0.189 (0.088) 0.157 (0.058)

0.176 - 0.173

9 VSALQKMGVRVQ 0.003 0.006 (0.060) 0.15 (0.063) (0.075) 0.176 (0.060) 0.164 (0.066) 0.163 (0.069)

0.149 0.157

10 MGVRVQNITGLA 0.36 0.23 (0.045) 0.118 (0.053) (0.095) 0.143 (0.034) 0.148 (0.069) 0.141 (0.080)

0.174 0.157

11 ADTRGILWAVE 0.001 0.11 (0.062) 0.16 (0.044) (0.049) 0.166 (0.037) 0.199 (0.097) 0.158 (0.046)

0.156 0.216

12 EAGSPAASAGVA 2.40E-02 0.015 (0.094) 0.236 (0.064) (0.041) 0.126 (0.046) 0.131 (0.028) 0.226 (0.053)

0.159 0.184

13 AVNRGRVASVEE 0.49 0.12 (0.055) 0.128 (0.069) (0.074) 0.172 (0.044) 0.171 (0.074) 0.160 (0.075)

0.165 0.166

14 VN GRVASVEEL 0.003 0.26 (0.062) 0.132 (0.070) (0.072) 0.166 (0.036) 0.170 (0.076) 0.152 (0.072)

HSP60 0.156 0.175

15 YHHSRIELSDP 6.00E-04 0.87 (0.058) 0.126 (0.072) (0.076) 0.167 (0.049) 0.172 (0.079) 0.155 (0.077)

0.156 0.165

16 IELSDPFERIGV 0.001 3.5 (0.051) 0.127 (0.078) (0.068) 0.172 (0.046) 0.164 (0.067) 0.149 (0.073)

17 FERIGVYFARSL 8.00E-04 7.1 0.171 0.153 (0.035) 0.169 0.194 (0.050) 0.194 (0.11) 0.162 (0.045)

(0.055) (0.052)

0.283 0.322

RSLAKRIHKRHA 0.002 0.32 (0.014) 0.243 (0.15) (0.18) 0.229 (0.095) 0.244 (0.13) 0.289 (0.17)

0.170 0.155

19 ADGVISSVILLR 0.01 0.065 (0.062) 0.129 (0.069) (0.062) 0.146 (0.036) 0.170 (0.073) 0.144 (0.064)

0.180 0.162

20 LLRAFLKASIPF 0.003 0.44 (0.060) 0.133 (0.079) (0.068) 0.164 (0.035) 0.171 (0.073) 0.150 (0.072)

0.161 0.158

21 LKASIPFIDQGL 0.003 0.86 (0.061) 0.123 (0.074) (0.082) 0.168 (0.033) 0.162 (0.077) 0.143 (0.079)

0.155 0.170

22 ASIPFIDQGLSP 0.003 0.86 (0.055) 0.150 (0.099) (0.085) 0.165 (0.031) 0.174 (0.084) 0.161 (0.089)

0.164 0.167

23 ASALASQKEAVC 0.014 3.5 (0.052) 0.137 (0.059) (0.075) 0.175 (0.052) 0.173 (0.074) 0.154 (0.069)

0.159 0.162

24 AYLHSHSFLLKD 0.001 2.4 (0.051) 0.130 (0.065) (0.061) 0.176 (0.036) 0.170 (0.081) 0.149 (0.063)

0.164 0.176

25 DASKVLGLIRS 0.014 2.4 (0.058) 0.139 (0.076) (0.064) 0.185 (0.045) 0.176 (0.069) 0.160 (0.070)

0.164 0.160

26 LIRSHLPDPLIG 0.001 1.7 (0.057) 0.137 (0.092) (0.076) 0.147 (0.034) 0.161 (0.078) 0.150 (0.082)

0.176 0.177

27 GEAFAEAVAYTG 0.008 0.17 (0.063) 0.134 (0.077) (0.084) 0.167 (0.038) 0.184 (0.091) 0.159 (0.082)

0.172 0.170

28 VAYTGHEGAVAL 0.008 5 (0.075) 0.123 (0.072) (0.081) 0.155 (0.038) 0.181 (0.099) 0.150 (0.079)

0.173 0.173

29 SQRSGSTLHLFC 0.93 0.62 (0.059) 0.132 (0.075) (0.070) 0.159 (0.031) 0.180 (0.092) 0.155 (0.073)

0.168 0.165

30 TLHLKGIQTQKG 0.01 1.2 (0.063) 0.128 (0.078) (0.079) 0.180 (0.041) 0.171 (0.080) 0.149 (0.078)

0.166 0.170

31 TQKGYRVPSFFP 6.00E-04 2.4 (0.055) 0.142 (0.043) (0.083) 0.176 (0.036) 0.183 (0.082) 0.158 (0.069)

0.168 0.169

32 FPHDSFHENF V 6.00E-04 0.44 (0.058) 0.129 (0.065) (0.082) 0.180 (0.064) 0.138 (0.079) 0.152 (0.076)

0.157 0.173

33 NPIVAPKIFVTD 1.00E-04 1.7 (0.056) 0.133 (0.069) (0.067) 0.146 (0.076) 0.197 (0.086) 0.155 (0.069)

0.16 0.154

34 FVTDQKIHCLFP 0.001 1.2 (0.057) 0.131 (0.059) (0.069) 0.146 (0.038) 0.166 (0.094) 0.144 (0.064)

0.169 0.170

35 DHAfflNAEDETS 0.001 2.4 (0.060) 0.138 (0.076) (0.069) 0.178 (0.045) 0.179 (0.072) 0.156 (0.071)

0.265 0.274

36 ETSRKLLKKRKH 0.003 1.7 (0.108) 0.222 (0.083) (0.15) 0.195 (0.069) 0.224 (0.105) 0.252 (0.123)

37 RKHRLENSIAH 0.001 0.32 0.155 0.126 (0.045) 0.146 0.169 (0.047) 0.173 (0.080) 0.138 (0.055)

(0.049) (0.060)

0.152 0.149

38 SIAIEPVKQDTA 0.004 1.2 (0.055) 0.13 (0.05) (0.052) 0.168 (0.040) 0.19 (0.092) 0.141 (0.053)

0.163 0.168

33 QDTAPLHELALK 0.003 0.87 (0.051) 0.133 (0.076) (0.081) 0.174 (0.050) 0.191 (0.086) 0.153 (0.079)

0.170 0.167

40 ALKTLNSTQESG 0.014 0.091 (0.045) 0.142 (0.069) (0.080) 0.179 (0.055) 0.187 (0.081) 0.157 (0.075)

0.178 0.176

41 ESGFVLGGGAAL 0.038 0.54 (0.064) 0.149 (0.074) (0.067) 0.186 (0.03) 0.197 (0.081) 0.165 (0.069)

0.163 0.141

42 AALLYATQSLSS 0.02 1.7 (0.062) 0.110 (0.063) (0.055) 0.146 (0.036) 0.179 (0.055) 0.128 (0.059)

0.180 0.174

43 LSSSPEHSQEEQ 0.003 0.62 (0.064) 0.140 (0.079) (0.089) 0.165 (0.045) 0.190 (0.097) 0.160 (0.084)

0.173 0.177

44 EEQAAVQILQTA 0.004 0.048 (0.062) 0.143 (0.063) (0.076) 0.163 (0.041) 0.224 (0.087) 0.163 (0.071)

0.167 0.170

45 DKLCSLGTPSLG 0.008 0.026 (0.058) 0.121 (0.065) (0.087) 0.155 (0.043) 0.189 (0.084) 0.149 (0.080)

0.199 0.231

46 YGPAYSSSSKDF 5.00E-04 2.3 (0.072) 0.245 (0.032) (0.099) · 0.202 (0.087) 0.209 (0.052) 0.237 (0.077)

0.208 0.250

47 VFSSPPFSNKPP 3.00E-04 0.86 (0.14) 0.300 (0.042) (0.14) 0.157 (0.043) 0.339 (0.039) 0.277 (0.097)

0.116 0.093

48 SLSSSPEHSQEE 1.00E-03 0.23 (0.030) 0.105 (0.019) (0.024) 0.111 (0.038) 0.097 (0.014) 0.098 (0.022)

Ct443 0.107 0.106

49 VDRKEVAPVHES 5.00E-04 12 (0.047) 0.119 (0.04) (0.048) 0.190 (0.24) 0.069 (0.006) 0.111 (0.045)

0.193 0.276

50 PVSFSGPTKGTIT 5.00E-04 12 (0.109) 0.253 (0.070) (0.17) 0.186 (0.093) 0.222 (0.068) 0.267 (0.13)

0.140 0.109

51 LTVPVSDTENTH 5.00E-04 0.033 (0.042) 0.110 (0.031) (0.041) 0.163 (0.079) 0.185 (0.033) 0.110 (0.036)

Ct381 0.136 0.104

52 VGIGVASDRPAL 0.001 0.11 (0.034) 0.114 (0.029) (0.040) 0.164 (0.075) 0.153 (0.057) 0.108 (0.036)

OmpB 0.127 0.125

53 AWSSGSDNELA 5.00E-04 2 (0.039) 0.137 (0.026) (0.057) 0.178 (0.089) 0.170 (0.063) 0.130 (0.046)

Cpn 0.060 0.063

54 EHFSPEPPNEPL Cp0236 2.30E-01 5.00E-05 (0.0073) 0.063 (0.012) (0.008) 0.068 (0.011) 0.061 (0.006) 0.063 (0.072)

0.057 0.062 0.059 0.0099

55 GSSLRTKEGNΉ 2.2 0.002 (0.0052) 0.060 (0.006) (0.011) (0.0076) 0.056 (0.005) (0.022)

Table 2: Performance of the Peptide 11 ELISA

Comparison Specificity (95% Sensitivity (95% Odds Ratio (95% confidence interval) confidence interval) confidence interval)

Performance of peptide 11 ELISA with a cutoff of 0.296 for absorbance at 450nm.

Sequelae vs Negative or 0.95 (0.75-1.00) 0.46 (0.19-0.74) 16.3 (1.65 - 160) Negative C.

pneumoniae

Multiple vs Negative or 0.95 (0.75 - 1.00) 0.176 (0.038 - 0.434) 4.07 (0.38 - 43.4) Negative C.

pneumoniae

Acute vs Negative or 0.95 (0.75-1.00) 0.14 (0.04-0.32) 3.17 (0.326 - 30.7) Negative C.

pneumoniae

Performance of peptide 11 ELISA with a cutoff of 0.408 for absorbance at 450nm.

Acute vs Sequelae 0.96 (0.81-1.00) 0.23 (0.05-0.53) 8.10 (0.75-87.2)

Multiple vs Acute 0.96 (0.73-1.00) 0.00 (0.00-0.338) 1.29 (0.024-69.6)

Table 3: Comparison of women attending the Sarfdarjung Hospital Gyneacology clinic who were positive in the peptide 11 assay by infertility, cervicitis, and current C. trachomatis infection status

.Cervicitis Infertility C trachomatis Peptide 11 Peptide 11

pcr/dfa status¹ negative positive

+ - + 3 0

15 0 primary - 25 0

+ primary - 24 7 primary + 33 2 secondary - 11 0 secondary + 9 0

C. trachomatis infection status was assessed by positive reactions in both PCR and DFA. Fisher's Exact Test for count data shows a significant difference in cohort proportions between peptide 11 ELISA un/reactive cohorts (P = 0.039).

Table 4: Initial Peptide Screen cohort results

Neg -Chronic >0.015 Neg vs NegCp <0.015 Chronic vs Acute >0.01

Peptide No. Neg Chronic Difference 95% CIofdiff. Neg NegCp Difference 95% CI ofdiff. Chronic Acute Difference 95% CI ofdiff.

1 0.1757 0.1717 -0.00406 -0.1260 to 0.1178 0.1757 0.1453 . -0.03048 -0.1671 to 0.1062 0.1717 0.1693 -0.00233 -0.1400 to 0.1353

2 0.1515 0.157 0.005545 -0.1163 to 0.1274 0.1515 0.1226 -0.02883 -0.1655 to 0.1078 0.157 0.169 0.012 -0.1256 to 0.1496

3 0.1648 0.1714 0.006598 -0.1153 to 0.1285 0.1648 0.139 -0.02582 -0.1625 to 0.1109 0.1714 0.1832 0.01175 -0.1259 to 0.1494

4 0.1668 0.1687 0.001848 -0.1200 to 0.1237 0.1668 0.1401 -0.02669 -0.1634 to 0.1100 0.1687 0.164 -0.00467 -0.1423 to 0.1330

5 0.1678 0.1633 -0.00449 -0.1264 to 0.1174 0.1678 0.126 -0.04182 -0.1785 to 0.09485 0.1633 0.1763 0.013 -0.1246 to 0.1506

6 0.1598 0.1548 -0.00499 -0.1269 to 0.1169 0.1598 0.1264 -0.03344 -0.1701 to 0.1032 0.1548 0.1632 0.008333 -0.1293 to 0.1460

7 0.1791 0.1688 -0.01034 -0.1322 to 0.1116 0.1791 0.1339 -0.04522 -0.1819 to 0.09145 0.1688 0.1653■ -0.00342 -0.1411 to 0.1342

8 0.1705 0.1528 -0.0178 -0.1397 to 0.1041 0.1705 0.1393 -0.0313 -0.1680 to 0.1054 0.1528 0.1885 0.03575 -0.1019 to 0.1734 *

9 0.1732 0.1763 0.003152 -0.1187 to 0.1250 0.1732 0.15 -0.02318 -0.1599 to 0.1135 0.1763 0.1637 -0.01267 -0.1503 to 0.1250

10 0.157 0.1489 -0.00808 -O.1300 to 0.1138 0.157 0.1183 -0.03875 -0.1754 to 0.09792 0.1489 0.1477 -0.00125 -0.1389 to 0.1364

11 0.1573 0.1738 0.01648 -0.1054 to 0.1384 • 0.1573 0.16 0.002727 -0.1339 to 0.1394 * 0.1738 0.199 0.02525 -0.1124 to 0.1629 * bHtrA 0.2161 0.1556 -0.06049 -0.1824 to 0.06140 * 0.2161 0.2358 0.01963 -0.1170 to 0.1563 0.1556 0.131 -0.02464 -0.1623 to 0.1130 *

12 0.1835 0.159 -0.02455 -0.1464 to 0.09735 * 0.1835 0.1284 -0.05517 -0.1918 to 0.08150 0.159 0.1708 0.01183 -0.1258 to 0.1495

13 0.1664 0.1645 -0.00186 -0.1238 to 0.1200 0.1664 0.1316 -0.03474 -0.1714 to 0.1019 0.1645 0.1698 0.005333 -0.1323 to 0.1430

14 0.1752 0.1559 -0.01927 -0.1412 to 0.1026 * 0.1752 0.1261 -0.04906 -0.1857 to 0.08761 0.1559 0.1723 0.01642 -0.1212 to 0.1541 *

15 0.1645 0.1564 -0.00813 -0.1300 to 0.1138 0.1645 0.1271 -0.03742 -0.1741 to 0.09925 0.1564 0.1643 0.007917 -0.1297 to 0.1456

16 0.1694 0.1709 0.001553 -0.1203 to 0.1234 0.1694 0.1529 -0.01649 -0.1532 to 0.1202 0.1709 0.1937 0.02275 -0.1149 to 0.1604 *

17 0.3218 0.2828 -0.03898 -0.1609 to 0.08291 • 0.3218 0.2428 -0.07907 -0.2157 to 0.05760 0-2828 0.2442 -0.03867 -0.1763 to 0.09897 •

18 0.1546 0.1699 - 0.01528 -0.1066 to0.1372 * 0.1546 0.1285 -0.02614 -0.1628 to 0.1105 0.1699 0.1698 -8.3E-05 -0.1377 to 0.1376

19 0.1624 0.1798 0.01747 -0.1044 to 0.1394 * 0.1624 0.1331 -0.02924 -0.1659 to 0.1074 0.1798 0.171 -0.00883 -0.1465 to 0.1288

20 0.1577 0.1613 0.003606 -0.1183 to 0.1255 0.1577 0.1229 -0:03485 -0.1715 to 0.1018 0.1613 0.162 0.000667 -0.1370 to 0.1383

21 0.1697 0:1553 -0.01439 -0.1363 to 0.1075 0.1697 0.1498 -0.01998 -0.1566 to 0.1167 0.1553 0.1735 0.01817 -0.1195 to 0.1558 *

22 0.167 0.164 -0.003 -0.1249 to 0.1189 0.167 0.1365 -0.0305 -0.1672 to 0.1062 0.164 _,0.1725 0.0085 -0.1291 to 0.1461

23 0.1625 0.1588 -0.00371 -0.1256 to 0.1182 0.1625 0.1304 -0.03208 -0.1687 to 0.1046 0.1588 0.1698 0.01108 -0.1266 to 0.1487

24 0.1758 0.1643 -0.01157 -0.1335 to 0.1103 0.1758 0.1386 -0.03719 -0.1739 to 0.09948 0.1643 0.1762 0.01192 -0.1257 to 0.1496

25 0.1596 0.1637 0.00403 -0.1179 to 0.1259 0.1596 0.1371 -0.02251 -0.1592 to 0.1142 0.1637 0.1612 -0.0025 -0.1401 to 0.1351

26 0.1769 0.1757 -0.00124 -0.1231 to 0.1207 0.1769 0.1336 -0.04328 -0.1800 to 0.09338 0.1757 0.1837 0.008 -0.1296 to 0.1456

27 0.1696 0.1718 0.002197 -0.1197 to 0.124I 0.16% 0.1234 -0.04626 -0.1829 to 0.09041 0.1718 0.181 0.009167 -0.1285 to 0.1468

28 0.1728 0.1731 0.000265 -0.1216 to 0.1222 0.1728 0.1315 -0.04132 -0.1780 to 0.09535 0.1731 0.1798 0.00675 -0.1309 to 0.1444

29 0.1649 0.1682 0.003258 -0.1186 to 0.1252 0.1649 0.1278 -0.03716 -0.1738 to 0.09951 0.1682 0.1712 0.003 -0.1346 to 0.1406

30 0.1699 0.166 -0.00391 -0.1258 to 0.1180 0.1699 0.1424 -0.02753 -0.1642 to 0.1091 0.166 0.183 0.017 -0.1206 to 0.1546

31 0.169 0.1678 -0.00125 -0.1231 to 0.1206 0.169 0.1289 -0.04013 -0.1768 to 0.09654 0.1678 0.138 -0.02975 -0.1674 to 0.1079

32 0,173 0.1573 -0.01567 -0.1376 to 0.1062 0.173 0.1318 -0.04125 -0.1779 to 0.09542 0.1573 0.1972 0.03983 -0.09780 to 0.1775

33 0.1538 0.1575 0.003682 -0.1182 to 0.1256 0.1538 0.1309 -0.02294 -0.1596 to 0.1137 0.1575 0.1658 0.008333 -0.1293 to 0.1460

34 0.1695 0.1693 -0.0003 -0.1222 to 0.1216 0.1695 0.1379 -0.03167 -0.1683 to0.1050 0.1693 0.1787 0.009417 -0.1282 to 0.1471

35 0.2736 0.2653 -0.0083 -0.1302 to 0.1136 0.2736 0.222 -0.05164 -0.1883 to 0.08503 0.2653 0.2237 -0.04167 -0.1793 to 0.09597

36 0.146 0.1545 0.0085 -0.1134 toO.1304 0.146 0.1263 -0.01975 -0.1564 to 0.1169 0.1545 0.1727 0.01817 -0.1195 to0.1558

37 0.1493 0.1519 0.002644 -0.1192 to 0.1245 0.1493 0.13 -0.01927 -0.1559 to 0.1174 0.1519 0.19 0.03808 -0.09955 to 0.1757

38 0.1679 0.1634 -0.00449 -0.1264 to 0.1174 0.1679 0.1333 -0.03466 -0.1713 to 0.1020 0.1634 0.191 0.02758 -0.1101 to 0.1652

39 0.1671 0.1697 0.002576 -0.1193 to 0.1245 0.1671 0.1423 -0.02484 -0.1615 to 0.1118 0.1697 0.1873 0.01767 -0.1200 to 0.1553

40 0.1764 0.1775 0.001136 -0.1208 to0.1230 0.1764 0.1489 -0.02749 -0.1642 to 0.1092 0.1775 0.1968 0.01933 -0.1183 to 0.1570

41 0.1408 0.163 0.02218 -0.09971 to 0.1441 0.1408 0.1096 -0.03119 -0.1679 to 0.1055 0.163 0.1788 0.01583 -0.1218 to 0.1535

42 0.1741 0.1798 0.005659 -0.1162 to 0.1276 0.1741 0.1403 -0.03384 -0.1705 to 0.1028 0.1798 0.1897 0.009917 -0.1277 to 0.1476

43 0.1774 0.173 -0.00436 -0.1263 to 0.1175 0.1774 0.1433 -0.03411 -0.1708 to 0.1026 0.173 0.2238 0.05083 -0.08680 to 0.1885

44 0.1697 0.1674 -0.00231 -0.1242 to 0.1196 0.1697 0.121 -0.04873 -0.1854 to 0.08794 0.1674 0.1885 0.02108 -0.1166 to 0.1587

HSP60-b2 0.231 0.1984 -0.03256 -0.1544 to 0.08934 0.231 0.2453 0.01429 -0.1224 to 0.1510 * 0.1984 0.209 0.01056 -0.1271 to 0.1482

HSP60-E2 0.2495 0.2079 -0.0416 -0.1635 to 0.08029 0.2495 0.3001 0.05064 -0.08603 to 0.1873 0.2079 0.3388 0.1309 -0.006786 to 0.2685 h2 0.0925 0.1.158 0.0233 -0.09859 to 0.1452 0.0925 0.105 0.0125 -0.1242 to 0.1492 * 0.1158 0.0965 -0.0193 -0.1569 to 0.1183

443-a3 0.1058 0.1073 0.001455 -0.1204 to 0.1233 0.1058 0.1191 0.01332 -0.1233 to 0.1500 * 0.1073 0.06933 -0.03794 -0.1756 to 0.09970

443-n2 0.2758 0.1926 -0.08324 -0.2051 to 0.03865 0.2758 0.2534 -0.02237 -0.1590 to 0.1143 0.1926 0.2223 0.02969 -0.1079 to 0.1673

443-q 0.1091 0.1397 0.03056 -0.09134 to 0.1524 0.1091 0.1101 0.001032 -0.1356 to 0.1377 • 0.1397 0.185 0.04533 -0.09230 to 0.1830

381-e2 0.1044 0.1362 0.03184 -0.09006 to 0.1537 0.1044 0.1143 0.009922 -0.1267 to 0.1466 * 0.1362 0.1532 0.017 -0.1206 to 0.1546 ompA-B 0.1248 0.1268 0.002015 -0.1199 to 0.1239 0.1248 0.1365 0.01168 -0.1250 to 0.1484 * 0.1268 0.1703 0.0435 -0.09414 to 0.1811

CplO 0.0625 0.06039 -0.002111 -0.1221 toO.1179 0.0625 0.06331 0.0008125 -0.1337 to 0.1353 0.06039 0.06063 0.0002361 -0.1352 to 0.1357

Cpll 0.06238 0.05711 -0.005264 -0.1252 to 0.1147 0.06238 0.05963 -0.002750 -0.1373 to O.1318 0.05711 0.0560 -0.001111 -0.1366 to 0.1344

Table 5: Cohort characterization using peptide 11 and Medac commercial kit

Table 7: Diagnostic performance of peptide 11 to detect C.trachomatis-related TFI in cohorts 1, 2 and 3

TFI positive (using TFI negative

MEDAC assay) (using MEDAC

assay)

Study Peptide Peptide Peptide Peptide Sensitivity Specificity P Odds

11 11 11 11 (95% CI) (95% CI) (X²) ratio positive negative positive negative (95%CI)

Samoan 49 10 57 106 0.83 [0.71,0.9 0.65 [0.57,0.7 O.001 9.11 [4.29, cohort 2] 2] 19.34]

Monash 2 0 4 26 1 [0.09,1] 0.87[0.69,0.9 0.005 29.44[1.2 cohort 6] 0,719.88]

Pregnancy 1 0 1 26 1[0.01,1] 0.96[0.81,1] 0.002 53[1.45,1 cohort 936.97] Table 8: Diagnostic performance of Medac MOMP+Medac cHSP60 to detect Ctrachomatis-related TFI in cohorts 1, 2 and 3

Table 9: Specificities and sensitivities of individual peptides and peptide combinations in identifying patients with tr c matis-related infertility (n=9 TFI+; n=18 TFI-)

TFI + TFI- Sensitivity - Specificity P Odds ratio

[95% CI] [95% CI1 (X²) [95%]

Assay Assay Assay Assay Assay

+ +

Peptide 11 ( 0.296) 6 3 2 16 0.67[0.3,0.93] 0.89[0.65,0.99] 0.003 16[2.12,120.65]

Peptide cHSP60 (> 0.25) 8 1 3 15 0.89 [0.52,1] 0.83[0.59,0.96] <0.001 40[3.56,450]

Peptide 443-n2 (>0.22) 9 0 2 16 1[0.55,1] 0.89[0.65,0.99] <0.001 125.4[5.43,2895.

88]

Peptide l l/chsp60 combo 9 0 1 17 1 [0.55,1] 0.94[0.73,1] <0.001 221.67[8.20,598 assay (>0.28) 9.75]

Peptidel l/chsp60/443-n2 7 2 3 15 0.78[0.4,0.97] 0.83[0.59,0.99] 0.002 17.5[2.36,129.51 (>0.23) ]

Peptidel 1 (>0.23) and 9 0 3 15 1 [0.55,1] 0.78[0.52,0.94] <0.001 61.22[2.95,1272. cHSP60 ( 0.22) 36]

Peptide 11 (0.18) and 443- 9 0 4 14 1[0.55,1] 0.78[0.52,0.94] <0.001 61.22[2.95,1272. n2 (>0.22) 36] cHSP60 (>0.22) and 443n2 9 0 2 16 1 [0.55,1] 0.89[0.65,0.99] <0.001 125.4[5.43,2895. (>0.22) 88]

Peptidel 1 (>0.18) and 9 0 2 16 1[0.55,1] 0.89[0.65,0.99] <0.001 125.4[5.43,2895. cHSP60 (>0.22) and 443n2 88]

(>0.22) Table 10: Overall inter and intra assay variability for peptide combination for 27 samples span of 6 days

Table 11: The physical and chemical properties of peptide 11, hsp60-e2 and 443-n2

* N-terminal biotin and C-terminal amide

I

Table 12: Pe tide 11 variants and their chemical properties

Table 13: SEQUENCE LISTING SUMMARY

SEQ ID NO Peptide or protein

1 Peptide 11

2 Fragment of Peptide 11

3 Fragment of Peptide 11

4 Fragment of Peptide 11

5 Variant of Peptide 11

6 Variant of Peptide 11

7 Peptide 47 (hsp60-e2)

8 Peptide 48 (hsp60)

9 Peptide 49 (ct443-a3)

10 Peptide 50 (ct443-n2)

11 Peptide 51 (ct443)

12 Peptide 52 (ct381)

13 Fragment of peptide 47

14 Fragment of peptide 48

, 15 Fragment of peptide 51

16 Fragment of peptide 52

17 HtrA amino acid sequence

18-66 Peptides 1-10, 12-46 and 53-55

67 GSGS- Peptide 11

Claims

An isolated peptide consisting essentially of the amino acid sequence ADTRGILWAVE (SEQ ID NO:l), or a variant or fragment thereof. The isolated peptide of Claim 1, wherein the fragment consists essentially of the amino acid sequence ADTRGILW (SEQ ID NO:2); ADTRGIL (SEQ ID NO:3) or TRGILVV (SEQ ID NO:4).

The isolated peptide of Claim 1, wherein the variant consists essentially of the amino acid sequence GIL AVEAGSP (SEQ ID NO:5) or ILVVAVEAGSPA (SEQ ID NO:6).

An isolated protein comprising one or a plurality of the amino acid sequence ADTRGILWAVE (SEQ ID NO:l) and/or one or a plurality of variants or fragments thereof, wherein the isolated protein further comprises no more than 450 amino acids of the amino acid sequence set forth in SEQ ID NO: 17.

An isolated protein comprising one or a plurality of the amino acid sequence ADTRGILWAVE (SEQ ID NO:l) and/or one or a plurality of variants or fragments thereof, wherein the isolated protein further comprises an amino acid sequence heterologous to the amino acid sequence set forth in SEQ ID NO: 17.

The isolated protein of Claim 4 or Claim 5, wherein the fragment consists essentially of the amino acid sequence ADTRGILW (SEQ ID NO:2); ADTRGIL (SEQ ID NO:3) or TRGILW (SEQ ID NO:4).

The isolated protein of any one of Claims 4-6, wherein the variant consists essentially of the amino acid sequence GILWAVEAGSP (SEQ ID NO:5) or ILWAVEAGSPA (SEQ ID NO:6).

The isolated protein of any one of Claims 4-7, further comprising one or a plurality of amino acid sequences set forth in any one of SEQ ID NOS:7- 16 and/or one or a plurality of variants or fragments thereof.

The isolated protein of Claim 8, wherein the amino acid sequence is set forth in any one of SEQ ID NOS:7, 9 or 10.

A method of determining whether a subject has been exposed to, or infected by, C. trachomatis, said method including the step of determining whether a biological sample obtained from a subject comprises one or more antibodies that binds one or more isolated peptides or proteins that comprise an isolated peptide, f agment or variant according to any one of Claims 1-3 or an isolated protein according to any one of Claims 4-9, wherein if the biological sample comprises the one or more antibodies the subject has been exposed to, or infected by, C. trachomatis.

A method of determining whether a subject has, or is predisposed to, one or more C. trachomatis sequelae and/or a chronic C. trachomatis infection, said method including the step of determining whether a biological sample obtained from a subject comprises one or more antibodies that binds one or more isolated peptides or proteins that comprise the isolated peptide according to any one of Claims 1-3 or the isolated protein according to any one of Claims 4-9, wherein if the biological sample comprises the one or more antibodies the subject has, or is predisposed to, the one or more C. trachomatis sequelae and/or chronic C. trachomatis infection.

The method of Claim 10 or Claim 11, wherein the one or more isolated peptides further comprise respective amino acid sequences according to any one of SEQ ID NOS:7-16.

The method of any one of Claims 10-12, wherein at least one of the one ore more antibodies binds an isolated peptide or protein accirding to any one of Claims 1-9.

The method of Claim 11, Claim 12 or Claim 13, wherein the C trachomatis sequelae include tubal factor infertility, ectopic pregnancy and/or pelvic inflammatory disease (PID).

The method of any one of Claims 10-14, wherein the sample is a serum sample.

The method of any one of Claims 10-15, wherein the step of determining whether the biological sample obtained from the subject comprises the antibody is performed by an immunoassay.

The method of Claim 16, wherein the immunoassay is an ELISA.

The method of Claim 17, wherein the immunoassay is a multiwell ELISA comprising a plurality of said isolated peptides or proteins, wherein each of said peptides or proteins is in a separate well. The method of any one of Claims 10-18, wherein the subject is a human female.

An isolated antibody which binds the isolated peptide, fragment or variant according to any one of Claims 1-3.

A composition or kit comprising the isolated peptide, variant or fragment according to any one of Claims 1-3, or the isolated protein according to any one of Claims 4-9 and one or more reagents that facilitate detection of an antibody that binds said peptide, fragment or variant. The composition or kit of Claim 21, which is an ELISA composition or kit.

An isolated nucleic acid comprising a nucleotide sequence encoding the isolated peptide, fragment or variant of any one of Claims 1-3 or the isolated protein of any one of Claims 4-9.

A genetic construct comprising the isolated nucleic acid of Claim 23 in combination with one or more other nucleotide sequences.

A host cell comprising the genetic construct of Claim 24.