CHLAMYDIA ANTIGENS AND USES THEREOF

TECHNICAL FIELD

[0001] The present invention relates generally to Chlamydia antigens, nucleic acids encoding said antigens and uses thereof. In particular, the present invention relates to pharmaceutical compositions and diagnostic reagents. In one form, the present invention relates to a vaccine against Chlamydia.

BACKGROUND

[0002] Chlamydiae are obligate intracellular bacteria that develop inside eukaryotic cells within membrane -bound vacuoles termed inclusions. The bacterium has a unique biphasic life cycle alternating between an extracellular infectious form (elementary body; EB) and an intracellular replicative form (reticulate body; RB). The bacterium only replicates inside cells, which is a key factor in its ability to remain hidden from a host's immune response and to cause persistent infections (Hogan R.J. et al. 2004).

[0003] In humans, Chlamydia trachomatis is a major infectious cause of human genital and eye disease. The bacterium is the most prevalent sexually transmitted bacterial infection worldwide with 1,401,906 infections reported in the USA in 2013 (CDC, 2014). It is also the single most important infectious agent associated with blindness with approximately 84 million people worldwide suffering C. trachomatis eye infections and 8 million people blinded as a result of the infection (WHO, 2012).

[0004] Ocular infections of C. trachomatis can cause trachoma, a chronic follicular conjunctivitis that results in scarring and blindness. The infection is transmitted through contact with eye and nose discharge of infected people, particularly young children who are a principal reservoir of infection (Mabey D.C., et al. 2003). It is also spread by flies which have been in contact with the eyes and noses of infected people (Mabey D.C., et al. 2003). Newborns can also develop a Chlamydia eye infection from an infected mother during vaginal childbirth (Frommell, G.T., et al.1979; Hammerschlag, M.R., et al. 1982; Heggie, A.D., et al. 1981 ; Schachter, J., et al. 1986).

[0005] Genital infections of C. trachomatis can cause cervicitis in women and urethritis and proctitis in both men and women. The infection is typically transmitted through sexual contact with the penis, vagina, mouth or anus of an infected partner and is commonly caused by C. trachomatis serovars D-K. The majority of infections remain localised in the genital tract and often only cause mild inflammation or are asymptomatic. However, without symptoms, many affected individuals remain untreated and the infection can progress to serious upper genital tract complications, such as, e.g., pelvic inflammatory disease (PID), tubal factor infertility, ectopic pregnancy and chronic pelvic pain.

[0006] Lymphogranuloma venereum (LGV) is another type of sexually transmitted disease (STD) caused by C. trachomatis serovars LI, L2 and L3 and often resulting from dissemination of the infection in the genital tract of women. LGV is a disease of the lymphatic tissue that occurs commonly in the developing world, and has most recently emerged as a cause of outbreaks of proctitis among men who have sex with men (MSM) (O'Farrell, N., et al. 2008; White, J.A., 2009).

[0007] In contrast to C. trachomatis, many of the eight other currently described species in the genus Chlamydia can infect multiple host species.

[0008] For example, C. precorum is a widespread pathogen of economically important livestock species such as cattle, sheep, goats and pigs. In livestock, C. precorum infections manifest as a range of diseases such as polyarthritis, pneumonia, conjunctivitis and encephalomyelitis, while also being linked to diseases of the gastrointestinal and urogenital tracts (Fukishi, H., et al. 1992; Polkinghorne, A., et al. 2009). While C. precorum infections in livestock are of economic concern to primary producers globally, the best example of the pathogenic potential of this bacterium is its infection of the koala, a native Australian marsupial, and the resulting debilitating ocular and urogenital tract diseases (Jackson, M., et al. 1999; McColl, K.A., et al. 1984). In this capacity, C. precorum is a key threatening process in the long-term survival of this native species (Polkinghorne, A., et al. 2013).

[0009] While antibiotic therapy effectively eliminates chlamydial infections, it does not always affect established pathology and can adversely effect animal gut microbial flora. Also, the presence of asymptomatic infections in both human and animal chlamydial infections makes control of infection by treatment of symptomatic individuals alone unlikely to succeed. Furthermore, if left untreated, chlamydial infections are more likely to lead to chronic inflammatory conditions with the severity of the disease related to the persistence of infection or frequency of reinfection. Accordingly, prevention of chlamydial infections by vaccination is an attractive alternative.

[0010] Previous vaccines against Chlamydia comprised live or attenuated pathogens. However, a disadvantage in using live vaccines is the risk of vaccine induced infection. Also, studies have shown that live or attenuated pathogen based vaccines provide only short lived protection, and that subjects immunised with insufficient antigen can suffer a hypersensitivity reaction upon re- exposure to Chlamydia.

[0011] An alternative to live or attenuated pathogen vaccines is randomly assessing specific antigens of Chlamydia for immunisation. However, this approach is limited as there is no assurance that antibodies produced in response to such an antigen will provide protection against subsequent infection by the pathogen. Consequently, often a large number of antigens must be assessed before an antigen that is protective against disease is found. Some disadvantages of conventional vaccines were overcome with the development of "genetic immunisation " (Tang et al. 1992), wherein host cells are inoculated with plasmid DNA encoding a pathogen protein.

[0012] Furthermore, vaccine studies have shown that in addition to a strong antibody response, ongoing protection from Chlamydia infections is also dependent on a strong cell mediated immune response (Kollipara A., et al. 2013).

[0013] One promising vaccine antigen candidate is major outer membrane protein (MOMP), which is encoded by the ompA gene (Kaltenboeck, B., et al. 1993). MOMP is highly immunogenic in both humans and animals and thus has been studied in detail as a vaccine candidate. MOMP also accounts for 60% of the bacterium outer membrane (Sun, G., et al. 2007). However, a major problem with MOMP-based vaccines comes from the sequence diversity present in its surface - exposed variable domains. For example, in human genital chlamydial infections, MOMP immunisation from one serovar will only elicit protection against the same or closely related serovars (Batteiger, B. 1996). Furthermore, sequence analyses of the surface-exposed variable domains encoded by C. precorum MOMP-encoding ompA from geographically distinct wild koala populations has revealed significant diversity (10-30%) (Carey, A.J., et al. 2010; Kindrachuk, J., et al. 2009; Miyairi, I., et al. 2010).

[0014] Accordingly, there is a need for a Chlamydia vaccine antigen candidate that is capable of providing long lasting and broad spectrum protection against Chlamydia spp.

SUMMARY OF INVENTION

[0015] The present inventor has identified Chlamydia peptide antigens that may be useful in diagnosis, treatment and/or prevention of Chlamydia infections, particularly C. precorum and C. trachomatis infections.

[0016] In one aspect of the present invention, there is provided an isolated, recombinant or synthetic peptide comprising, consisting of, or consisting essentially of an amino acid sequence sharing at least 75% sequence identity with an amino acid sequence as set forth in any one of SEQ ID NOs: l-47, or a fragment, variant, derivative, mimetic or pharmaceutically acceptable salt thereof.

[0017] In some embodiments, the amino acid sequence of the isolated, recombinant or synthetic peptide may share at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% sequence identity with an amino acid sequence as set forth in any one of SEQ ID NOs:l-47, or a fragment, variant, derivative, mimetic or pharmaceutically acceptable salt thereof.

[0018] In preferred embodiments, the amino acid sequence of the isolated, recombinant or synthetic peptide may share at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% sequence identity with an amino acid sequence as set forth in any one of SEQ ID NOs:l-12 a fragment, variant, derivative, mimetic or pharmaceutically acceptable salt thereof.

[0019] In more preferred embodiments, the amino acid sequence of the isolated, recombinant or synthetic peptide may share at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence as set forth in any one of SEQ ID NOs: l-4 or a fragment, variant, derivative, mimetic or pharmaceutically acceptable salt thereof.

[0020] In even more preferred embodiments, the amino acid sequence of the isolated, recombinant or synthetic peptide may share at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% sequence identity with an amino acid sequence as set forth in any one of SEQ ID NOs: l-4 or a fragment, variant, derivative, mimetic or pharmaceutically acceptable salt thereof.

[0021] In most preferred embodiments, the isolated, recombinant or synthetic peptide has an amino acid sequence as set forth in any one of SEQ ID NOs: l-4 or a fragment, variant, derivative, mimetic or pharmaceutically acceptable salt thereof.

[0022] In another aspect of the present invention, there is provided an isolated, recombinant or synthetic peptide comprising, consisting of, or consisting essentially of an amino acid sequence as set forth in any one of SEQ ID NOs:l-47 containing at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions, or a fragment, variant, derivative, mimetic or pharmaceutically acceptable salt thereof.

[0023] In preferred embodiments, the amino acid sequence of the isolated, recombinant or synthetic peptide may be as set forth in any one of SEQ ID NOs:l-12 containing at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions, or a fragment, variant, derivative, mimetic or pharmaceutically acceptable salt thereof.

[0024] In more preferred embodiments, the isolated, recombinant or synthetic peptide comprises an amino acid sequence as set forth in any one of SEQ ID NOs:l-4 containing at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions, or a fragment, variant, derivative, mimetic or pharmaceutically acceptable salt thereof.

[0025] In some embodiments, the isolated, recombinant or synthetic peptide or fragment, variant, derivative, mimetic or pharmaceutically acceptable salt thereof may be an immunogenic peptide, fragment, variant, derivative, mimetic or pharmaceutically acceptable salt. An immunogenic peptide, fragment, variant, derivative, mimetic or pharmaceutically acceptable salt is capable of eliciting an immune response in a subject administered the peptide, fragment, variant, derivative, mimetic or pharmaceutically acceptable salt.

[0026] In other embodiments, the isolated, recombinant or synthetic peptide or fragment, variant, derivative, mimetic or pharmaceutically acceptable salt thereof may be a haptenic peptide, fragment, variant, derivative, mimetic or pharmaceutically acceptable salt. A haptenic peptide, fragment, variant, derivative, mimetic or pharmaceutically acceptable salt is capable of reacting with cognate antibodies, but cannot itself elicit an immune response.

[0027] In yet another aspect of the present invention, there is provided an isolated, recombinant or synthetic protein comprising, consisting of, or consisting essentially of one or more of the isolated, recombinant or synthetic peptide or a fragment, variant or derivative thereof as hereinbefore described, wherein the isolated protein does not include a full length MOMP amino acid sequence, such as set forth in any one of SEQ ID NOs:48-62.

[0028] In some embodiments, the isolated, recombinant or synthetic protein may include a heterologous amino acid sequence.

[0029] A "fragment" of the isolated, recombinant or synthetic peptide or protein of the invention is a peptide or protein with an amino acid sequence that constitutes less than 100%, but at least 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% of the amino acid sequence of the isolated, recombinant or synthetic peptide or protein. In some embodiments, the fragment may include as little as 10%, 5% or 3% of the entire isolated, recombinant or synthetic protein. In some embodiments, a fragment may also include sub -fragments, e.g., sub-fragments of identified fragments. Fragments of the isolated, recombinant or synthetic peptide or protein of the invention may be immunogenic or haptenic, preferably immunogenic.

[0030] A "variant" of the isolated, recombinant or synthetic peptide or protein of the invention is a peptide or protein in which one or more amino acids have been replaced by different amino acids. It is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing or at least reducing the immunogenic properties of the peptide or protein (i.e., conservative substitutions). Variants of the isolated, recombinant or synthetic peptides or proteins of the invention may include homologous and orthologous peptides and proteins.

[0031] A "derivative" of the isolated, recombinant or synthetic peptide or protein of the invention is a peptide or protein that has been altered, for example, by conjugation or complexing with a chemical moiety, by post-translational modification (including, but not limited to, phosphorylation, glycosylation, acetylation, lipidation or pegylation), or by the addition of one or more amino acids (including, for example, the addition of a protein or tag to assist purification). The incorporation of non-naturally occurring amino acids is also encompassed by "derivative", such as, e.g., where one or more side groups of naturally occurring a-amino acids have been modified.

[0032] A "mimetic" of the isolated, recombinant or synthetic peptide or protein of the invention is a peptide or protein including chemical amino acid analogues/derivatives. The mimetic may be conformationally constrained molecules or alternatively molecules which are not conformationally constrained such as, for example, non-constrained peptide sequences. The term "conformationally constrained molecules" means conformationally constrained peptides and conformationally constrained peptide analogues and derivatives.

[0033] As mentioned, the peptides of the present invention may be in the form a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable salts may include, but are not limited to, salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, lactic, citric, benzoic and glutamic acids. Non-pharmaceutically acceptable salts may also fall within the scope of the invention as these may be useful as intermediates in the preparation of pharmaceutically acceptable salts or during storage or transport.

[0034] According to another aspect of the present invention, there is provided an isolated, recombinant or synthetic nucleic acid encoding the peptide or the protein of the invention, including a fragment thereof.

[0035] As used herein, the term "nucleic acid" designates single or double-stranded mRNA, RNA, cRNA and DNA, said DNA being inclusive of cDNA and genomic DNA. A nucleic acid may be native or recombinant and may include one or more artificial nucleotides, e.g., nucleotides not normally found in nature. Nucleic acid encompasses modified purines (e.g., inosine, methylinosine and methyladenosine) and modified pyrimidines (e.g., thiouridine and methylcytosine).

[0036] Fragments of isolated, recombinant or synthetic nucleic acids of the invention may include a nucleotide sequence that constitutes less than 100% of the nucleic acid sequence of the invention, for example, less than or equal to 99%, 98%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 6%, 4%, 2% or even 1%. It will be appreciated that a fragment may include all integer values less than 100%, for example, the percentage values as set forth above and others. A fragment may include a polynucleotide, an oligonucleotide, a probe, a primer and an amplification product, e.g., a PCR product. A fragment may also include a sub-fragment, i.e., a fragment of a fragment such as a primer derived from a larger fragment of the invention.

[0037] A "probe" may be a singular or double-stranded oligonucleotide or polynucleotide, suitably labelled for the purposes of detecting complementary sequences in Northern or Southern blotting, for example. Generally, a "polynucleotide" may be a nucleic acid having eighty (80) or more contiguous nucleotides, while an "oligonucleotide" may have less than eighty (80) contiguous nucleotides.

[0038] A "primer" usually may be a single-stranded oligonucleotide having between 5 and 200 contiguous nucleotides, including all integer values therebetween, e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180 and 190, and which is capable of annealing to a complementary nucleic acid (i.e., "template") and being extended in a template-dependent fashion by the action of a DNA polymerase, such as, e.g., Taq polymerase, RNA-dependent DNA polymerase or Sequenase™.

[0039] For example, primers may be used to amplify nucleic acids specifically for a selected strains or species to thereby determine a presence of said strains or species of an organism, preferably a Chlamydia spp., as will be discussed in detail later.

[0040] Primers may also be used to amplify nucleic acids common to one or more strains or species. Such primers are an example of primers that are preferably used to detect one or more strains or species of an organism, preferably a Chlamydia spp., most preferably C trachomatis or C. precorum, as will be discussed in detail later.

[0041] The present invention may also involve combination therapies, such as the administration to a subject of an isolated, recombinant or synthetic peptide, protein or nucleic acid encoding the peptide or protein of the invention, including a fragment, variant, derivative, mimetic or pharmaceutically acceptable salt thereof together with other agents or procedures for treating or preventing a Chlamydia infection or a disease or disorder associated with or caused by a Chlamydia infection. For example, the agent may be in the form of an immunotherapeutic composition, vaccine or antigen presenting cell loaded or pulsed with an antigen. The antigen presenting cell may be loaded or pulsed with antigen by contacting the cell with an antigen, for example the isolated, recombinant or synthetic peptide or protein, fragment, variant, derivative, mimetic or pharmaceutically acceptable salt of the invention. The antigen presenting cell may be, for example, a dendritic cell.

[0042] The isolated, recombinant or synthetic peptides and proteins or isolated, recombinant or synthetic nucleic acids encoding the peptides and proteins of the invention, including any fragment, variant, derivative, mimetic or pharmaceutically acceptable salt thereof, may be used as pharmaceuticals. Consequently in a further aspect of the present invention, there is provided a pharmaceutical composition for preventing or ameliorating a Chlamydia infection, said composition including at least one peptide, protein or nucleic acid of the invention, including a fragment, variant, derivative, mimetic or pharmaceutically acceptable salt thereof. The pharmaceutical composition may include a pharmaceutically acceptable carrier or excipient.

[0043] In a preferred form, the pharmaceutical composition comprises a plurality of peptides, proteins and/or nucleic acids of the invention, including fragments, variants, derivatives, mimetics or pharmaceutically acceptable salts thereof. It will be appreciated that a pharmaceutical composition comprising a plurality of antigens may provide an improved immune response against the pathogen, namely one or more forms of Chlamydia. Accordingly, a pharmaceutical composition preferably comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-five, thirty or more different peptides, proteins and/or nucleic acids of the invention, including fragments, variants, derivatives, mimetics or pharmaceutically acceptable salts thereof.

[0044] The carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the composition and not being deleterious to the subject. [0045] Pharmaceutical compositions include those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal or parenteral (including intramuscular, sub-cutaneous, intrathecal and intravenous) administration or in a form suitable for administration by inhalation or insufflation. The pharmaceutical composition may be especially suitable for parenteral administration. The isolated, recombinant or synthetic peptide or protein of the present invention, the isolated, recombinant or synthetic nucleic acid encoding the peptide or protein or a fragment, variant, derivative, mimetic or pharmaceutically acceptable salt thereof may be placed with a pharmaceutically acceptable carrier or excipient in a pharmaceutical composition. The composition may be in the form of a solid (including tablets, filled capsules, powders, capsules, suppositories, dispersible granules and pessaries), or a liquid (including solutions, suspensions, emulsions, colloids, elixirs, creams, gels and foams). In one embodiment, the pharmaceutical composition may be in the form of a sterile injectable solution for parenteral use.

[0046] The nature of the pharmaceutical composition and the carrier or excipient will depend on the route of administration and the nature of the condition and the subject being treated. It is believed that the choice of a particular carrier or delivery system, and route of administration could be readily determined by a person skilled in the art. In some circumstances it may be necessary to protect the isolated, recombinant or synthetic peptide or protein of the present invention including any fragment, variant, derivative, mimetic or pharmaceutically acceptable salt thereof (in view of the amide bonds) by means known in the art, for example, by micro encapsulation. The route of administration should also be chosen such that the isolated, recombinant or synthetic peptide or protein or the isolated, recombinant or synthetic nucleic acid encoding the peptide or protein, or a fragment, variant, derivative, mimetic or pharmaceutically acceptable salt thereof, reaches its site of action.

[0047] The pharmaceutically acceptable carrier or excipient may be either a solid or a liquid. A solid carrier or excipient may act as a diluent, flavouring agent, solubilizer, lubricant, suspending agent, binder, preservative, tablet disintegrating agent or an encapsulating material. Suitable solid carriers and excipients would be known to a skilled person.

[0048] If the pharmaceutical composition is a powder, the active component and a carrier or excipient may both be finely divided powders which are mixed together.

[0049] If the pharmaceutical composition is a tablet, the active component may be mixed with a suitable amount of a carrier or excipient which has the necessary binding capacity before compaction into a tablet of the desired shape and size. [0050] Exemplary carriers or excipients for powders and tablets may include, for example, magnesium carbonate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, a low melting wax, cocoa butter and the like.

[0051] Liquid form preparations may include, for example, water or water -propylene glycol solutions. For example, parenteral injection liquid preparations can be formulated as solutions in aqueous polyethylene glycol solution.

[0052] Liquid pharmaceutical compositions may be formulated in unit dose form. For example, the compositions may be presented in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers. Such compositions may include a preservative. The compositions may also include formulatory agents such as suspending, stabilising and/or dispersing agents. The composition may also be in powder form for constitution with a suitable vehicle (such as sterile water) before use. Liquid carriers and excipients may include colorants, flavours, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, suspending agents and the like.

[0053] Aqueous solutions for oral use may be prepared by dissolving the active compound in water and adding colourants, thickeners, flavours, and stabilizing agents, as necessary.

[0054] Aqueous suspensions for oral use may be prepared by dispersing the active component in water with viscous material, such as natural or synthetic gums, resins, methyl cellulose or other suspending agents.

[0055] The pharmaceutical composition may also include solid-form preparations intended to be converted into a liquid form for oral administration. For topical administration to the epidermis, the compounds may be formulated as an ointment, cream or lotion, or as a transdermal patch.

[0056] For oral administration, the active compound may be incorporated with carriers or excipients and used in the form of ingestible tablets, buccal or sublingual tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like. Some of these oral administration routes may have the potential to avoid liver metabolism.

[0057] The compositions may also be administered by inhalation in the form of an aerosol spray from a pressurised dispenser or container, which contains a propellant such as carbon dioxide gas, dichlorodifluoromethane, nitrogen, propane or other suitable gas or gas combination.

[0058] In some embodiments, the pharmaceutical composition is an immunotherapeutic composition capable of eliciting an immune response in a subject administered the pharmaceutical composition. Preferably, the immunotherapeutic composition is a vaccine. The vaccine may be a prophylactic vaccine and/or a therapeutic vaccine. In such an embodiment, the composition may further include an adjuvant, such as, e.g., aluminium hydroxide, aluminium hydroxyphosphate or any other suitable immunostimulating adjuvant. Suitable adjuvants include, but are not limited to adjuvants for use in animals or humans, for example, SBAS2, SBAS4, QS21 or ISCOMs.

[0059] In one such embodiment, the immunotherapeutic composition or vaccine when administered to a subject may be capable of providing protective immunity in said subject against one or more species, biovar, serovar and/or genotypes of Chlamydia.

[0060] Preferably, the pharmaceutical composition when administered to a subject is capable of preventing or treating a Chlamydia infection, reducing the severity of a Chlamydia infection, reducing symptoms associated with a Chlamydia infection or ameliorate an infection caused by one or more species, biovar, serovar and/or genotypes of Chlamydia.

[0061] Generally, the infection comprises infection of the gastrointestinal tract, the rectal tract, the urogenital tract or the eyes of a subject.

[0062] Preferably, the composition is capable of treating or preventing a disease or disorder associated with or caused by a Chlamydia infection.

[0063] The disease or disorder may be selected from the group consisting of atherosclerosis, sexually transmitted disease, urethritis, epididymitis, cervicitis, pelvic inflammatory disease, ectopic pregnancy, infertility, tubal factor infertility, epididymitis, proctitis, reactive arthritis, conjunctivitis including neonatal conjunctivitis, mucopurulent cervicitis, rupture of membranes, premature delivery, cervical carcinoma, stenosis in an infected organ and inflammation, polyarthritis, pneumonia and encephalomyelitis.

[0064] In another aspect, the present invention provides a method of inducing an immune response in a subject, said method including administering to the subject an effective amount of a pharmaceutical composition of the invention or at least one isolated, recombinant or synthetic peptide, protein or nucleic acid encoding the peptide or protein of the invention, including a fragment, variant, derivative, mimetic of the invention or a pharmaceutically acceptable salt thereof.

[0065] In a further aspect, the present invention provides a method of treating or preventing a Chlamydia infection in a subject, said method including administering to the subject an effective amount of a pharmaceutical composition of the invention or at least one isolated, recombinant or synthetic peptide, protein or nucleic acid encoding the peptide or protein of the invention, including a fragment, variant, derivative, mimetic of the invention or a pharmaceutically acceptable salt thereof.

[0066] In yet a further aspect, the present invention provides a method of treating a disease or disorder associated with or caused by a Chlamydia infection in a subject, said method including administering to the subject an effective amount of a pharmaceutical composition of the invention or at least one isolated, recombinant or synthetic peptide, protein or nucleic acid encoding the peptide or protein of the invention, including a fragment, variant, derivative, mimetic of the invention or a pharmaceutically acceptable salt thereof.

[0067] In yet another aspect, the present invention provides use of a pharmaceutical composition of the invention or at least one isolated, recombinant or synthetic peptide, protein or nucleic acid encoding the peptide or protein of the invention, including a fragment, variant, derivative, mimetic of the invention or a pharmaceutically acceptable salt thereof to prevent infection, reduce severity of infection, reduce symptoms caused by or improve recovery from infection by a Chlamydia spp. in a subject, preferably C trachomatis or C precorum.

[0068] As used herein, the terms "treatment" and "prevention" are to be considered in their broadest contexts. For example, the term "treatment" does not necessarily imply that a subject is treated until full recovery. The term "treatment" includes amelioration of the symptoms of a disease or disorder, or reducing the severity of a disease or disorder. Similarly, "prevention" does not necessarily imply that a subject will never contract a disease or disorder. "Prevention" may be considered as reducing the likelihood of onset of a disease or disorder, or preventing or otherwise reducing the risk of developing a disease or disorder.

[0069] An "effective amount" of a pharmaceutical composition of the invention or an isolated, recombinant or synthetic peptide, protein or nucleic acid encoding the peptide or protein of the invention, including a fragment, variant, derivative, mimetic of the invention or a pharmaceutically acceptable salt thereof means an amount necessary to at least partly attain a desired response, or to delay the onset or progression of the disease or disorder being treated. The amount may vary depending on factors such as: the health and physical condition of the subject whom is being treated, the taxonomic group of the subject being treated, the extent of treatment/prevention desired, the formulation of the composition, and the assessment of the medical situation. It is expected that the "effective amount" will fall within a broad range that can be determined through routine trials.

[0070] As used herein, the term "subject" may include mammals, especially humans, primates, livestock animals, laboratory test animals, companion animals and wild animals (whether captive or free) such as, e.g., koalas. Livestock animals may include sheep, cattle, oxen, buffalos, pigs, horses and donkeys. Laboratory test animals may include mice, rabbits, rats, pigs, koalas and guinea pigs. Companion animals may include dogs and cats. In one embodiment, the subject is a human. In another embodiment, the subject is a livestock animal, such as e.g., sheep, cattle, pigs and/or horses. In yet another embodiment, the subject is a marsupial, such as, e.g., a koala.

[0071] Another aspect of the present invention provides an isolated antibody or fragment thereof that binds to at least one isolated, recombinant or synthetic peptide or protein of the invention, including a fragment, variant, derivative, mimetic or pharmaceutically acceptable salt thereof. The antibody may be a polyclonal or monoclonal antibody, preferably a monoclonal antibody. The fragment may be a binding fragment and may include Fc or Fab fragments of a polyclonal or monoclonal antibody referred to above. The antibody may also include single chain Fc antibodies (scFvs).

[0072] In another aspect, the present invention provides a method of detecting one or more species, biovar, serovar and/or genotypes of Chlamydia in a biological sample, said method including the steps of:

combining with the biological sample at least one isolated, recombinant or synthetic peptide or protein of the invention, including a fragment, variant, derivative, mimetic of the invention or a pharmaceutically acceptable salt thereof; and

determining the presence of an antibody or fragment thereof bound to said peptide, protein or fragment, variant, derivative, mimetic or pharmaceutically acceptable salt thereof, wherein the presence of said antibody or fragment thereof is indicative of the presence of said one or more species, biovar, serovar and/or genotypes of Chlamydia in the biological sample.

[0073] In a further aspect of the present invention, there is provided a method of diagnosing an infection of a subject with one or more species, biovar, serovar and/or genotypes of Chlamydia, or absence of an infection, said method including the steps of:

contacting a biological sample obtained from said subject with at least one isolated, recombinant or synthetic peptide or protein of the invention, including a fragment, variant, derivative, mimetic of the invention or a pharmaceutically acceptable salt thereof; and

determining the presence or absence of a complex between said peptide, protein or fragment, variant, derivative, mimetic or pharmaceutically acceptable salt thereof and Chlamydia- specific antibodies in said sample, wherein the presence of said complex is indicative of said infection. [0074] In yet a further aspect of the present invention, there is provided a kit for detecting Chlamydia in a biological sample or diagnosing a Chlamydia infection in a subject, said kit including at least one isolated, recombinant or synthetic peptide or protein of the invention, including a fragment, variant, derivative, mimetic of the invention or a pharmaceutically acceptable salt thereof, and/or antibodies capable of binding said peptide, protein or fragment, variant, derivative, mimetic or pharmaceutically acceptable salt thereof.

[0075] In another aspect of the present invention, there is provided use of peptides encoding the epitopes 4, 28, 41 and 42 from MOMP protein as herein described, including any fragments, variants, derivatives, mimetics or pharmaceutically acceptable salts thereof, or a composition comprising the peptides or fragments, variants, derivatives, mimetics or pharmaceutically acceptable salts thereof for: eliciting an immune response from a subject administered said peptides, fragments, variants, derivatives, mimetics or pharmaceutically acceptable salts thereof or said composition; preventing infection, reducing severity of infection, reducing symptoms caused by or improving recovery from infection by a Chlamydia spp. in a subject; diagnosing an infection of a subject with one or more species, biovar, serovar and/or genotype of Chlamydia; or detecting one or more species, biovar, serovar and/or genotype of Chlamydia in a biological sample.

[0076] In yet another aspect, the present invention provides a kit for detecting Chlamydia in a biological sample or diagnosing a Chlamydia infection in a subject, said kit including one or more peptides encoding the epitopes 4, 28, 41 and 42 from MOMP protein as herein described, including any fragments, variants, derivatives, mimetics or pharmaceutically acceptable salts thereof and/or antibodies of fragments thereof capable of binding said peptides or fragments, variants, derivatives, mimetics or pharmaceutically acceptable salts thereof.

[0077] Throughout this specification, preferred aspects and embodiments apply, as appropriate, separately, or in combination, to other aspects and embodiments, mutatis mutandis, whether or not explicitly stated as such.

[0078] The present invention will now be described further with reference to the following examples, which are illustrative only and non-limiting.

BRIEF DESCRIPTION OF DRAWINGS

[0079] Various embodiments of the invention will be described with reference to the following drawings, in which:

[0080] Figure 1: Shows antibody titres (IgG) against recombinant MOMP (rMOMP) and UV- inactivated whole chlamydial elementary bodies (EBs) in plasma from two animal groups sampled at 0, 2 and 6 month time points. In particular, (A) and (B) respectively show antibody titres (IgG) against rMOMP and EBs for non-vaccinated C. precorum PCR negative animals; and (C) and (D) respectively show antibody titres (IgG) against rMOMP and EBs for non-vaccinated C. precorum PCR positive animals.

[0081] Figure 2: Shows antibody titres (IgG) against recombinant MOMP (rMOMP) and UV- inactivated whole chlamydial elementary bodies (EBs) in plasma from two animal groups sampled at 0, 2 and 6 month time points and vaccinated thrice. In particular, (A) and (B) respectively show antibody titres (IgG) against rMOMP and EBs for C. precorum PCR negative animals; and (C) and (D) respectively show antibody titres (IgG) against rMOMP and EBs for non-vaccinated C. precorum PCR positive animals. Arrows indicate the vaccination time points.

[0082] Figure 3: Shows neutralising antibody titres expressed as fold change neutralisation in plasma from four animal groups sampled at 0, 2 and 6 month time points. In particular: (A) shows fold change neutralisation in plasma from non-vaccinated C. precorum PCR negative animals; (B) shows fold change neutralisation in plasma from vaccinated C. precorum PCR negative animals; (C) shows fold change neutralisation in plasma from non-vaccinated C. precorum PCR positive animals; and (D) shows fold change neutralisation in plasma from vaccinated C. precorum PCR positive animals. Arrows indicate the vaccination time points.

[0083] Figure 4: Shows the correlation between C. precorum infection load versus plasma antibody titres (IgG) against recombinant MOMP (rMOMP) for non-vaccinated C. precorum PCR positive animals. The infection loads were measured at the genital site. The spearman correlation coefficients were measure the relationship between IgG and infection load. The level of significance were measured as *p - 0.01-0.05, **p - 0.001-0.01, ***/><0.001.

[0084] Figure 5: Shows epitope directed antibody response profiles, measured as epitope specificity, for vaccinated and non-vaccinated C. precorum PCR negative and positive animals at 0 and 6 month time points. In particular: (A) shows epitope specificity for C. precorum PCR negative and PCR positive animals at the 0 month time point; and (B) shows epitope specificity for C. precorum PCR negative and PCR positive animals at the 6 month time point where some animals have been vaccinated. Specifically: (Bl) shows epitope specificity for non-vaccinated C. precorum PCR positive animals; (B2) shows epitope specificity for vaccinated C. precorum PCR positive animals (Tash, Bev, Oldbean, Popy and Fiona) and vaccinated C. precorum PCR negative animals (Robyn, Pepper, Maya, Hunky, Harry and Randall); and (B3) shows epitope specificity for non- vaccinated C. precorum PCR negative animals. The grey boxes indicate unique epitope response profiles for vaccinated C. precorum PCR positive animals.

[0085] Figure 6: Shows: (A) epitope directed antibody response profiles, measured as epitope specificity, for plasma sampled from vaccinated C. precorum PCR negative and positive animals before (left) and after (right) removal of antibodies against unique epitopes via absorption with peptides for the epitopes. The grey boxes indicate vaccine-induced unique epitope response profiles; (B) shows neutralising antibody titres expressed as fold change neutralisation of the pre- absorption plasma (left) and post-absorption plasma (right) in which antibodies to the unique epitopes have been removed. Results are expressed as the means + SD of five animals per group. Error bars represent SD.

KEY TO SEQUENCE LISTING

[0086] SEQ ID NO:l - Amino acid sequence of peptide 1 corresponding to epitope 4 of MOMP from C. precorum genotypes A, F and H;

[0087] SEQ ID NO:2 - Amino acid sequence of peptide 2 corresponding to epitope 28 of MOMP from C. precorum genotypes A, F, G and H;

[0088] SEQ ID NO:3 - Amino acid sequence of peptide 3 corresponding to epitope 41 of MOMP from C. precorum genotypes A, F and H;

[0089] SEQ ID NO:4 - Amino acid sequence of peptide 4 corresponding to epitope 44 of MOMP from C. precorum genotype A;

[0090] SEQ ID NO: 5 - Amino acid sequence of variant of peptide 1 from C. precorum genotype G;

[0091] SEQ ID NO: 6 - Amino acid sequence of variant of peptide 1 from C. trachomatis;

[0092] SEQ ID NO:7 - Amino acid sequence of variant of peptide 2 from C. trachomatis;

[0093] SEQ ID NO: 8 - Amino acid sequence of variant of peptide 3 from C. precorum genotype G;

[0094] SEQ ID NO:9 - Amino acid sequence of variant of peptide 3 from C. trachomatis;

[0095] SEQ ID NO: 10 - Amino acid sequence of variant of peptide 4 from C. precorum genotype F; [0096] SEQ ID NO: 11 - Amino acid sequence of variant of peptide 4 from C. precorum genotypes G and H;

[0097] SEQ ID NO: 12 - Amino acid sequence of variant of peptide 4 from C. trachomatis;

[0098] SEQ ID NO: 13 - Amino acid sequence of peptide 13 from C. precorum genotypes A, F, G and H;

[0099] SEQ ID NO: 14 - Amino acid sequence of peptide 14 from C. precorum genotypes A, F, G and H;

[00100] SEQ ID NO: 15 - Amino acid sequence of peptide 15 from C. precorum genotypes A, G and H;

[00101] SEQ ID NO: 16 - Amino acid sequence of variant of peptide 15 from C. precorum genotype F;

[00102] SEQ ID NO: 17 - Amino acid sequence of peptide 16 from C. precorum genotypes A, F, G and H;

[00103] SEQ ID NO: 18 - Amino acid sequence of peptide 17 from C. precorum genotypes A, F, G and H;

[00104] SEQ ID NO: 19 - Amino acid sequence of peptide 18 from C. precorum genotype A;

[00105] SEQ ID NO:20 - Amino acid sequence of variant of peptide 18 from C. precorum genotype F;

[00106] SEQ ID NO:21 - Amino acid sequence of variant of peptide 18 from C. precorum genotype G;

[00107] SEQ ID NO:22 - Amino acid sequence of variant of peptide 18 from C. precorum genotype H;

[00108] SEQ ID NO:23 - Amino acid sequence of peptide 19 from C. precorum genotypes A and H;

[00109] SEQ ID NO:24 - Amino acid sequence of variant of peptide 19 from C. precorum genotype F; [00110] SEQ ID NO:25 Amino acid sequence of variant of peptide 19 from C. precorum genotype G;

[00111] SEQ ID NO:26 Amino acid sequence of peptide 20 from C. precorum genotypes A, F, G and H;

[00112] SEQ ID NO:27 Amino acid sequence of peptide 21 from C. precorum genotypes A, F, G and H;

[00113] SEQ ID NO:28 Amino acid sequence of peptide 22 from C. precorum genotypes A and H;

[00114] SEQ ID NO:29 Amino acid sequence of variant of peptide 22 from C. precorum genotype F;

[00115] SEQ ID NO.30 Amino acid sequence of variant of peptide 22 from C. precorum genotype G;

[00116] SEQ ID NO:31 Amino acid sequence of peptide 23 from C. precorum genotypes A, F, G and H;

[00117] SEQ ID NO:32 Amino acid sequence of peptide 24 from C. precorum genotype A;

[00118] SEQ ID NO:33 Amino acid sequence of variant of peptide 24 from C. precorum genotype F;

[00119] SEQ ID NO:34 Amino acid sequence of variant of peptide 24 from C. precomm genotypes G and H;

[00120] SEQ ID NO:35 Amino acid sequence of peptide 25 from C. precorum genotype A;

[00121] SEQ ID NO:36 Amino acid sequence of variant of peptide 25 from C. precorum genotype F;

[00122] SEQ ID NO:37 Amino acid sequence of variant of peptide 25 from C. precorum genotypes G and H;

[00123] SEQ ID NO:38 Amino acid sequence of peptide 26 from C. precorum genotype A;

[00124] SEQ ID NO:39 Amino acid sequence of variant of peptide 26 from C. precorum genotype F;

[00125] SEQ ID NO:40 Amino acid sequence of variant of peptide 26 from C. precorum genotype G;

[00126] SEQ ID NO:41 Amino acid sequence of variant of peptide 26 from C. precorum genotype H;

[00127] SEQ ID NO:42 Amino acid sequence of peptide 27 from C. precorum genotype A;

[00128] SEQ ID NO:43 Amino acid sequence of variant of peptide 27 from C. precorum genotype F;

[00129] SEQ ID NO:44 Amino acid sequence of variant of peptide 27 from C. precorum genotypes G and H;

[00130] SEQ ID NO:45 Amino acid sequence of peptide 28 from C. precorum genotype A;

[00131] SEQ ID NO:46 Amino acid sequence of variant of peptide 28 from C. precorum genotype F;

[00132] SEQ ID NO:47 Amino acid sequence of variant of peptide 28 from C. precorum genotypes G and H;

[00133] SEQ ID NO:48 Amino acid sequence of MOMP from C. precorum genotype A;

[00134] SEQ ID NO:49 Amino acid sequence of MOMP from C. precorum genotype F;

[00135] SEQ ID NO:50 Amino acid sequence of MOMP from C. precorum genotype G;

[00136] SEQ ID NO:51 Amino acid sequence of MOMP from C. precorum genotype H;

[00137] SEQ ID O:52 Amino acid sequence of MOMP from C. trachomatis;

[00138] SEQ ID NO:53 Amino acid sequence of MOMP from C. trachomatis serovar A;

[00139] SEQ ID NO:54 Amino acid sequence of MOMP from C. trachomatis serovar B;

[00140] SEQ ID NO:55 Amino acid sequence of MOMP from C. trachomatis serovar C;

[00141] SEQ ID NO:56 Amino acid sequence of MOMP from C. trachomatis serovar D; [00142] SEQ ID NO:57 - Amino acid sequence of MOMP from C. trachomatis serovar E;

[00143] SEQ ID NO:58 - Amino acid sequence of MOMP from C. trachomatis serovar F;

[00144] SEQ ID NO: 59 - Amino acid sequence of MOMP from C trachomatis serovar H; [00145] SEQ ID NO:60 - Amino acid sequence of MOMP from C. trachomatis serovar LI ; [00146] SEQ ID NO:61 - Amino acid sequence of MOMP from C trachomatis serovar L2; and [00147] SEQ ID NO:62 - Amino acid sequence of MOMP from C trachomatis serovar L3; DETAILED DESCRIPTION

[00148] The present invention has arisen, at least in part, from investigations into the development of a koala Chlamydia vaccine. Previous work by the inventor and others has shown that immunisation with recombinant MOMP (rMOMP) protein can lead to a strong antibody response, as well as a specific lymphocyte response, up to at least one year post vaccination (Kollipara, A., et al. 2012; Kollipara, A., et al. 2013; Carey, A.J., et al. 1989). This led to further investigations into the presence of antibodies in naturally infected koalas (C. precorum positive animals) and in koalas following vaccination. Specifically, investigations into antibodies produced by koalas naturally infected with Chlamydia, the level and specificity of these antibodies, and whether a vaccine can be developed capable of producing antibodies of a higher titre, or a different specificity, than antibodies produced from a natural Chlamydia infection and that are able to provide further protection against subsequent disease.

[00149] The above investigations led to the discovery that diseased koalas with a natural Chlamydia infection produced plasma antibodies that specifically recognised a unique set of rMOMP epitopes. This unique set of rMOMP epitopes were not recognised by the plasmas antibodies from non-diseased koalas with a natural Chlamydia infection (see Figure 5). Furthermore, rMOMP vaccinated koalas with a natural Chlamydia infection produced plasma antibodies that recognised a different unique set of rMOMP epitopes.

[00150] The above investigations also led to the discovery that in vitro neutralising antibody titres were highest in koalas that had a previous Chlamydia infection and were then administered the rMOMP vaccine (see Figure 3).

[00151] The above discoveries, in turn, led to the discovery that one or more of the plasma antibodies to the different unique set of rMOMP epitopes identified in rMOMP vaccinated koalas with a natural Chlamydia infection were responsible for the strong in vitro neutralisation ability seen in such koalas (see Figure 6).

[00152] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g. in molecular biology, biochemistry, structural biology, and/or computational biology). Standard techniques are used for molecular and biochemical methods (see generally, Sambrook et al., 2001, Ausubel et al. 1999 and Green and Sambrook, 2012, which are incorporated herein by reference) and chemical methods.

[00153] For the purposes of this invention, by "isolated" it is meant that the subject material has been removed from its natural state or otherwise has been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material includes material in native and recombinant form. For example, isolated Chlamydia spp. and nucleic acids, proteins and peptides isolated therefrom.

[00154] It will be appreciated that there has been some confusion over the taxonomic family Chlamydiaceae. In 1999, a new chlamydial taxonomy was proposed based on two genera: Chlamydia and Chlamydophila, and nine species (Everett, K.D.E., et al. 1999). However, this new taxonomy was not widely adopted. In 2011, the taxonomic confusion was finally resolved with the publication of Volume 4 of the 2 ^nd Edition of Bergey's Manual of Systematic Bacteriology, which described one species Chlamydia and nine species. Accordingly, as described herein, the taxonomic family Chlamydiaceae is made up of a single genera Chlamydia and the following nine species: Chlamydia trachomatis, C. pneumoniae, C. muridarum, C. suis, C. abortus, C. psittaci, C. caviae, C. felis and C. precorum. The different species are further divided into biovars and serovars based on disease pathology and serology, respectively. For example, C. trachomatis is divided into two biovars, the trachoma biovar, which incorporates serovars A, B, Ba, C, D, Da, E, F, G, H, I, la, J and K, and the lymphogranuloma venereum biovar that consists of serovars LI, L2 and L3. Likewise, PCR sequence analysis of the predominant animal pathogen C. precorum has revealed at least 13 different genotypes, including genotypes A, F, G and H.

[00155] Accordingly, use of the term "Chlamydia " herein generally relates to all the abovementioned species, biovars, serovars and genotypes unless specifically referred to otherwise.

[00156] In one form, the present invention relates to isolated, recombinant or synthetic peptides or proteins or isolated, recombinant or synthetic nucleic acids encoding the peptides or proteins of the invention, including any fragment, variant, derivative, mimetic or pharmaceutically acceptable salt thereof.

[00157] In another form, the present invention relates to pharmaceutical compositions, including immunotherapeutic compositions and vaccines, containing said peptides, proteins, nucleic acids or fragments, variants, derivatives, mimetics or pharmaceutically acceptable salts thereof for use: in inducing an immune response; treating or preventing infections caused by one or more species, biovar, serovar or genotypes of Chlamydia; or treating or preventing a disease or disorder associated with or caused by such an infection. In some embodiments, the pharmaceutical compositions of the invention may be for use in preventing such infections, reducing the severity of such infections, reducing symptoms caused by such infections or to improve recovery from such infections. In preferred embodiments, the Chlamydia spp., biovar, serovar and/or amino type is one capable of infecting a human or an animal, preferably a livestock animal such as, e.g., cattle, sheep, pig, goat, and/or horse.

[00158] In another form, the present invention relates to isolated antibodies and fragments thereof that bind to said peptides, proteins, nucleic acids or fragments, variants, derivatives, mimetics or pharmaceutically acceptable salts thereof.

[00159] In yet another form, the present invention relates to diagnostic methods and reagents for detecting one or more species, biovar, serovar and/or genotypes of Chlamydia. In a preferred form, the present invention relates to methods and reagents for detecting one or more animal Chlamydia spp., biovar, serovar and/or genotypes, such as e.g., C. precorum and C. pneumoniae. In another preferred form, the present invention relates to methods and reagents for detecting one or more human Chlamydia spp., biovar, serovar and/or genotypes, such as e.g., C. trachomatis.

[00160] By "endogenous" nucleic acid or protein, it is meant a nucleic acid or protein that may be found in a native organelle, cell, tissue, bacteria, organism or animal in isolation or otherwise.

Peptides and proteins

[00161] The terms "peptide" or "protein" refer to an amino acid polymer, comprising natural and/or non-natural amino acids, including L- and D-isomeric forms, as are well understood in the art. A polypeptide may contain more than 60 contiguous amino acids. A peptide may contain up to 60 contiguous amino acids.

[00162] As previously mentioned, a "fragment" of the isolated, recombinant or synthetic peptides or proteins of the invention is a peptide or protein having an amino acid sequence that constitutes less than 100% of an entire amino acid sequence of the isolated, recombinant or synthetic peptide or protein. A fragment preferably comprises less than 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 60%, 50%, 40%, 30% or 20% of the entire isolated, recombinant or synthetic peptide or protein. In some embodiments, the fragment may comprise as little as even 10%, 5% or 3% of the entire isolated, recombinant or synthetic protein. In some embodiments, a fragment may also include sub -fragments, for example, sub- fragments of identified fragments.

[00163] The fragment may comprise an immunogenic fragment or a haptenic fragment.

[00164] An immunogenic fragment refers to a fragment capable of inducing an immune response in an animal (or human), whether protective or not. It will be appreciated that an immunogenic fragment capable of inducing an immune response, in particular a B-cell response, may be useful in generating antibodies for uses including diagnostics, affinity purification, as actives in a pharmaceutical composition and as a research agent. An immune response also refers to a T-cell mediated response and a response involving antigen presenting cells.

[00165] It will be appreciated that based on the method of isolating the peptides of the invention, identified peptide or protein fragments will likely be immunogenic. Such fragments include peptides and proteins comprising an amino acid sequence indicated in the sequence listings and include both human and animal infecting forms of Chlamydia.

[00166] An immunogenic fragment in one form is a fragment capable of being bound by an antibody that binds to the same region of the full-length peptide or protein. The immunogenic fragment in another form is capable of binding an appropriate target ligand.

[00167] A haptenic fragment is a fragment which reacts with cognate antibodies, but cannot itself elicit an immune response. It can be conjugated with an immunogenic carrier. Useful carriers are well known in the art and include for example: thyroglobulin; albumins such as human serum albumin; toxins, toxoids or any mutant cross reactive material (CRM) of the toxin from tetanus, diphtheria, pertussis, Pseudomonas, E. coli, Staphylococcus, and Streptococcus; polyamino acids such as poly(lysine: glutamic acid); influenza; Rotavirus VP6, Parvovirus VP1 and VP2; hepatitis B virus core protein; hepatitis B virus recombinant vaccine and the like. Alternatively, a fragment or epitope of a carrier protein or other immunogenic protein may be used. For example, a haptenic fragment of the invention can be coupled to a T cell epitope of a bacterial toxin, toxoid or CRM. In this regard, reference may be made to U.S. Patent No 5,785,973 which is incorporated herein by reference. [00168] In some embodiments, a "fragment" is a small protein or peptide, for example of at least 6, preferably at least 10 and more amino acids in length, which comprises one or more antigenic determinants or epitopes. Larger fragments comprising one or more peptides or proteins are also contemplated.

[00169] Peptides, proteins and fragments thereof of the present invention may be obtained through the application of standard recombinant nucleic acid techniques or synthesized using conventional liquid or solid phase synthesis techniques. For example, reference may be made to solution synthesis or solid phase synthesis as described, for example, in Chapter 9 entitled "Peptide Synthesis " by Atherton and Shephard that is included in a publication entitled "Synthetic Vaccines " edited by Nicholson and published by Blackwell Scientific Publications. Alternatively, peptides or proteins can be produced by digestion of a protein or peptide of the invention with a suitable proteinase. The digested fragments can be purified by, for example, high performance liquid chromatographic (HPLC) techniques.

[00170] As also previously mentioned, a "variant" of the isolated, recombinant or synthetic peptides and proteins of the invention is a peptide or protein in which one or more amino acids have been replaced by different amino acids. It is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing or at least reducing the immunogenic properties of the peptide or protein (i.e., conservative substitutions). Such conservative substitutions are shown in Table 1 under the heading of "Exemplary Substitutions".

[00171] Substantial changes in function can be made by selecting substitutions that are less conservative or non-conservative as is known in the art. Generally, the substitutions which are likely to produce the greatest changes in a peptide or protein's properties are those in which: (a) a hydrophilic residue (e.g., Ser or Thr) is substituted with a hydrophobic residue (e.g., Leu, He, Phe or Val); (b) a cysteine or proline is substituted for, or by, any other residue; (c) a residue having an electropositive side chain (e.g., Arg, His or Lys) is substituted for, or by, an electronegative residue (e.g., Glu or Asp); or (d) a residue having a bulky side chain (e.g., Try or Phe) is substituted for, or by, one having a smaller side chain (e.g.,. Ser or Ala) or no side chain (e.g., Gly). A variant may also include one or more amino acid deletions and/or additions. [00172] Table 1 - Exemplary substitutions.

[00173] With regard to peptide and protein variants, these can be created by mutagenizing a peptide or protein or by mutagenizing an encoding nucleic acid, such as by random mutagenesis or site-directed mutagenesis. Examples of nucleic acid mutagenesis methods are provided in Chapter 9 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al, supra which is incorporated herein by reference.

[00174] It will be appreciated by the skilled person that site -directed mutagenesis is best performed where knowledge of the amino acid residues that contribute to the immunogenic properties is available. In many cases, this information is not available, or can only be inferred by molecular modelling approximations, for example.

[00175] In such cases, random mutagenesis is contemplated. Random mutagenesis methods include chemical modification of proteins by hydroxylamine (Ruan et al, 1997 Gene 188:35), incorporation of dNTP analogs into nucleic acids (Zaccolo et ai, 1996, J. Mol. Biol. 255:589) and PCR-based random mutagenesis such as described in Stemmer, 1994, Proc. Natl. Acad. Sci. USA 91:10747 or Shafikhani et ai, 1997, Biotechniques 23:304, each reference of which is incorporated herein. It is also noted that PCR-based random mutagenesis kits are commercially available, such as the Diversify™ kit (Clontech).

[00176] Again as previously mentioned, a "derivative" of the isolated, recombinant or synthetic peptides or proteins of the invention, include peptide and proteins that have been altered, for example, by conjugation or complexing with a chemical moiety, by post-translational modification, by the addition and/or deletion of one or more amino acids or by the incorporation of non-naturally occurring amino acids, such as, e.g., where one or more side groups of naturally occurring a-amino acids have been modified.

[00177] Thus, for example the amino acids may be replaced with a variety of uncoded or modified amino acids such as the corresponding D-amino acid or N-methyl amino acid. Other modifications may include replacing naturally occurring a-amino acids with chemically analogous structures. Examples include molecules containing gem-diaminoalkyl groups or alklylmalonyl groups. Yet further modifications include substitution of hydroxyl, thiol, amino and carboxyl functional groups with chemically similar groups.

[00178] Other examples of other unnatural amino acids or chemical amino acid analogues/derivatives which can be introduced as a substitution or addition include, but are not limited to, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t- butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogues in general.

[00179] "Addition" of amino acids may include fusion of a peptide, protein or variant thereof with other peptides or proteins. Particular examples of such peptides or proteins include amino (N) and carboxyl (C) terminal amino acids added for use as "tags".

[00180] N-terminal and C-terminal tags include known amino acid sequences which bind a specific substrate, or bind known antibodies, preferably monoclonal antibodies. pRSET B vector (ProBond™; Invitrogen Corp.) is an example of a vector comprising an N-terminal 6X-His-tag which binds ProBond™ resin. [00181] Other derivatives contemplated by the invention include modification to side chains, incorporation of unnatural amino acids and/or their derivatives during protein synthesis and the use of cross linkers and other methods which impose conformational constraints on the proteins, fragments and variants of the invention. Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by acylation with acetic anhydride; acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; amidination with methyl acetimidate; carbamoylation of amino groups with cyanate; pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH ₄ ; reductive alkylation by reaction with an aldehyde followed by reduction with NaBH ₄ ; and trinitrobenzylation of amino groups with 2, 4,6-trinitrobenzene sulphonic acid (TNBS).

[00182] The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, by way of example, to a corresponding amide.

[00183] The guanidine group of arginine residues may be modified by formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

[00184] Sulphydryl groups may be modified by methods such as performing acid oxidation to cysteic acid; formation of mercurial derivatives using 4-chloromercuriphenylsulphonic acid, 4- chloromercuribenzoate; 2-chloromercuri-4-nitrophenol, phenylmercury chloride, and other mercurials; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; carboxymethylation with iodoacetic acid or iodoacetamide; and carbamoylation with cyanate at alkaline pH.

[00185] Tryptophan residues may be modified, for example, by alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphonyl halides or by oxidation with N- bromosuccinimide.

[00186] Tyrosine residues may be modified by nitration with tetranitromethane to form a 3- nitrotyrosine derivative.

[00187] The imidazole ring of a histidine residue may be modified by N-carbethoxylation with diethylpyrocarbonate or by alkylation with iodoacetic acid derivatives.

[00188] Examples of incorporating unnatural amino acids and derivatives during protein synthesis include the use of 4-amino butyric acid, 6-aminohexanoic acid, 4-amino-3-hydroxy-5- phenylpentanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, t-butylglycine, norleucine, norvaline, phenylglycine, ornithine, sarcosine, 2-thienyl alanine and/or D-isomers of amino acids.

[00189] Also as previously described, a "mimetic" of the isolated, recombinant or synthetic peptides or proteins, include peptides and proteins including chemical amino acid analogues/derivatives. The mimetic may be conformationally constrained molecules or alternatively molecules which are not conformationally constrained such as, for example, non- constrained peptide sequences. The term "conformationally constrained molecules" means conformationally constrained peptides and conformationally constrained peptide analogues and derivatives.

[00190] The mimetic may be a peptidomimetic. A "peptidomimetic" is a molecule that mimics the biological activity or immunogenic properties of a peptide or even protein but is no longer peptidic in chemical nature. By strict definition, a peptidomimetic is a molecule that no longer contains any peptide bonds (i.e., amide bonds between amino acids). However, the term peptide mimetic is sometimes used to describe molecules that are no longer completely peptidic in nature, such as pseudo-peptides, semi-peptides and peptoids. Whether completely or partially non-peptide, peptidomimetics provide a spatial arrangement of reactive chemical moieties that closely resembles the three-dimensional arrangement of the peptides and proteins on which the peptidomimetic is based, including, if present, one or more antigenic determinants or epitopes. As a result of this similar geometry, the peptidomimetic preferably has a similar immunogenic effect as the immunogenic effect of the peptide or protein on which the peptidomimetic is based.

[00191] There are sometimes advantages for using a mimetic of a given peptide or protein rather than the peptide or protein itself, because peptides, including proteins, commonly exhibit two undesirable properties: (1) poor bioavailability; and (2) short duration of action. Peptide mimetics offer an obvious route around these two major obstacles, since the molecules concerned are small enough to be both orally active and have a long duration of action. There are also considerable cost savings and improved patient compliance associated with peptide mimetics, since they can be administered orally compared with parenteral administration for peptides or proteins. Furthermore, peptide mimetics are generally cheaper to produce than peptides or proteins.

[00192] Suitable peptidomimetics based on the isolated, recombinant or synthetic peptides or proteins of the invention can be developed using readily available techniques. Thus, for example, peptide bonds can be replaced by non-peptide bonds that allow the peptidomimetic to adopt a similar structure, and therefore immunogenic effect, to the original peptide or protein. Further modifications can also be made by replacing chemical groups of the amino acids with other chemical groups of similar structure.

[00193] Peptides and proteins in relation to the invention (inclusive of fragments, variants, and derivatives) may be prepared by any suitable procedure known to those of skill in the art.

[00194] For example, the peptide or protein may be prepared by a procedure including the steps of:

(i) preparing an expression construct which comprises a recombinant nucleic acid of the invention, operably linked to one or more regulatory nucleotide sequences, for example a T7 promoter;

(ii) transfecting or transforming the expression construct into a suitable host cell, for example E. coli; and

(iii) expressing the protein in said host cell.

[00195] Preferably, the recombinant nucleic acid of the invention encodes a peptide or protein of the invention, including a fragment, variant or homolog thereof. Preferred expression methods include those described in the examples.

[00196] Recombinant peptides and proteins may be conveniently expressed and purified by a person skilled in the art using commercially available kits, for example "ProBond™ Purification System" available from Invitrogen Corporation, Carlsbad, CA, USA, herein incorporated by reference. Alternatively, standard molecular biology protocols may be used, as for example described in Sambrook, et al, MOLECULAR CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989), incorporated herein by reference, in particular Sections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al, (John Wiley & Sons, Inc. 1995- 1999), incorporated herein by reference, in particular Chapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al, (John Wiley & Sons, Inc. 1995-1999) which is incorporated by reference herein, in particular Chapters 1, 5, 6 and 7.

Peptide, protein and nucleic acid sequence comparison

[00197] Terms used herein to describe sequence relationships between respective nucleic acids and peptides/proteins include "comparison window", "sequence identity", "percentage of sequence identity" and "substantial identity". Because respective nucleic acids/peptides/proteins may each comprise: (1) only one or more portions of a complete nucleic acid/peptide/protein sequence that are shared by the nucleic acids/peptides/proteins, and (2) one or more portions which are divergent between the nucleic acids/peptides/proteins, sequence comparisons are typically performed by comparing sequences over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of typically at least 6 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the respective sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (for example ECLUSTALW and BESTFIT provided by WebAngis GCG, 2D Angis, GCG and GeneDoc programs incorporated herein by reference) or by visual inspection by an operator and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.

[00198] The ECLUSTALW program is used to align multiple sequences. This program calculates a multiple alignment of nucleotide or amino acid sequences according to a method by Thompson, J. D., Higgins, D. G. and Gibson, T.J. (1994). This is part of the original ClustalW distribution, modified for inclusion in EGCG. The BESTFIT program aligns forward and reverse sequences and sequence repeats. This program makes an optimal alignment of a best segment of similarity between two sequences. Optimal alignments are determined by inserting gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman. ECLUSTALW and BESTFIT alignment packages are offered in WebANGIS GCG (The Australian Genomic Information Centre, Building J03, The University of Sydney, NSW 2006, Australia).

[00199] Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et ai, 1997, Nucl. Acids Res. 25 3389, which is incorporated herein by reference.

[00200] A detailed discussion of sequence analysis can be found in Chapter 19.3 of Ausubel et al, supra.

[00201] The term "sequence identity" is used herein in its broadest sense to include the number of exact nucleotide or 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. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For example, "sequence identity" may be understood to mean the "match percentage" calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA).

[00202] As generally used herein, a "homolog" shares a definable nucleotide or amino acid sequence relationship with a nucleic acid or protein of the invention as the case may be.

[00203] "Protein homologs" share at least 20%, preferably at least 75%, 80%, 85% or 90% and more preferably at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequences of proteins of the invention as hereinbefore described. It will be appreciated that a homolog comprises all integer values less than 100%, for example the percent value as set forth above and others.

[00204] Included within the scope of homologs are "orthologs", which are functionally-related proteins and their encoding nucleic acids, isolated from other organisms. For example, orthologs in relation to Chlamydia antigens include homologous proteins of animals known to be infectable by Chlamydia, including for example: mice, hamsters, swine, cattle, sheep, goats, birds, guinea pigs, cats, koalas and horses. An ortholog may comprise an amino acid sequence 100% identical with a protein from another species.

Nucleic acids

[00205] As previously described, the term "nucleic acid" as used herein designates single or double stranded mRNA, RNA1 cRNA and DNA, said DNA inclusive of cDNA and genomic DNA. A nucleic acid may be native or recombinant and may comprise one or more artificial nucleotides, e.g., nucleotides not normally found in nature. Nucleic acid encompasses modified purines (for example, inosine, methylinosine and methyladenosine) and modified pyrimidines (thiouridine and methylcytosine).

[00206] The term "isolated nucleic acid" as used herein refers to a nucleic acid subjected to in vitro manipulation into a form not normally found in nature. Isolated nucleic acid includes both native and recombinant (non-native) nucleic acids. For example, a nucleic acid isolated from a human, a koala, a livestock animal or the like.

[00207] A "polynucleotide" is a nucleic acid having eighty (80) or more contiguous nucleotides, while an "oligonucleotide" has less than eighty (80) contiguous nucleotides. [00208] In one embodiment, a nucleic acid "fragment" comprises a nucleotide sequence that constitutes less than 100% of a nucleic acid of the invention, for example, less than or equal to: 99%, 98%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 6%, 4%, 2% or even 1 %. It will be appreciated that a fragment comprises all integer values less than 100%, for example the percent value as set forth above and others. A fragment includes a polynucleotide, an oligonucleotide, a probe, a primer and an amplification product, e.g., a PCR product. A fragment also includes a sub-fragment, i.e., a fragment of a fragment such as a primer derived from a larger fragment of the invention.

[00209] A "probe" may be a single or double-stranded oligonucleotide or polynucleotide, suitably labeled for the purpose of detecting complementary sequences in Northern or Southern blotting, for example.

[00210] A "primer" is usually a single-stranded oligonucleotide, preferably having 20-50 contiguous nucleotides, which is capable of annealing to a complementary nucleic acid "template" and being extended in a template -dependent fashion by the action of a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or Sequenase™. For example, primers may be used to amplify nucleic acids specific for a selected strain or species to thereby determine a presence of said strain or species of an organism, preferably Chlamydia, as is discussed in more detail herein after.

[00211] Primers may be used to amplify nucleic acids common to one or more strains or species. Such primers are an example of primers that are preferably used to detect one or more strains or species of an organism, preferably Chlamydia, as is discussed in more detail herein after.

Variant nucleic acids

[00212] As used herein, the term nucleic acid "variant" means a nucleic acid of the invention for which the nucleotide sequence has been mutagenized or otherwise altered so as to encode substantially the same, or a modified peptide or protein. Such changes may be trivial, for example, in cases where more convenient restriction endonuclease cleavage and/or recognition sites are introduced without substantially affecting the immunogenic properties of an encoded peptide or protein when compared to a non-variant form. Other nucleotide sequence alterations may be introduced so as to modify the immunogenic properties of an encoded peptide or protein. These alterations may include deletion or addition of one or more nucleotide bases, or involve non- conservative substitution of one base for another. Such alterations can have profound effects upon the immunogenic properties of an encoded peptide or protein, possibly increasing or decreasing the immunogenic properties. In this regard, mutagenesis may be performed in a random fashion or by site-directed mutagenesis in a more "rational" manner. Standard mutagenesis techniques are well known in the art, and examples are provided in Chapter 9 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds Ausubel et al. (John Wiley & Sons NY, 1995), which is incorporated herein by reference.

[00213] For the purposes of host cell expression, the recombinant nucleic acid may be operably linked to one or more regulatory sequences in an expression vector, for example a T7 promoter.

[00214] 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. Examples of expression vectors include: eukaryotic expression vectors pC130, pC31 and pC32; and pRSET B (Invitrogen Corp.) and derivations thereof. An expression vector may include a bacterial expression vector as is commonly used to express recombinant proteins in bacteria, preferably for subsequent isolation of the recombinant protein. Such vectors include: pBEc-Q (Stratagene), pT7-FLAG-l (Sigma- Aldrich), TNT system (Promega) and the like.

[00215] By "operably linked" it is meant that said regulatory nucleotide sequence(s) is/are positioned relative to the recombinant nucleic acid of the invention to initiate, regulate or otherwise control transcription.

[00216] 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.

[00217] Typically, said one or more regulatory nucleotide sequences may include, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences.

[00218] Constitutive or inducible promoters as known in the art are contemplated by the invention. The promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter. For example, the lac promoter is inducible by IPTG.

[00219] The expression vector may further comprise a selectable marker gene to allow the selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used. For example, an ampicillin resistance gene for selection of positively transformed host cells when grown in a medium including ampicillin. [00220] The expression vector may also include a fusion partner (typically provided by the expression vector) so that the recombinant peptide or protein of the invention is expressed as a fusion protein with the fusion partner. An advantage of fusion partners is that they assist identification and/or purification of the fusion protein. Identification and/or purification may include using a monoclonal antibody or substrate specific for the fusion partner, for example a poly-His (for example 6X-His) tag or GST. A fusion partner may also comprise a leader sequence for directing secretion of a recombinant protein, for example an alpha-factor leader sequence.

[00221] Well known examples of fusion partners include poly-His or hexahistidine (6X-HIS)- tag, N-Flag, Fc portion of human IgG, glutathione-S-transferase (GST) and maltose binding protein (MBP), which are particularly useful for isolation of the fusion protein by affinity chromatography. For the purposes of fusion protein purification by affinity chromatography, relevant matrices for affinity chromatography may include nickel-conjugated or cobalt-conjugated resins, fusion protein specific antibodies, glutathione-conjugated resins, and amylose-conjugated resins respectively. Some matrices are available in a "kit" form, such as the ProBond™ Purification System (Invitrogene Corp.) which incorporates a 6X-His fusion vector and purification using ProBond™ resin.

[00222] In order to express the fusion protein, it is necessary to ligate a nucleic acid according to the invention into the expression vector so that the translational reading frames of the fusion partner and the nucleotide sequence of the invention coincide.

[00223] The fusion partners may also have protease cleavage sites, for example enterokinase (available from Invitrogen Corp. as EnterokinaseMax™), Factor X _a or Thrombin, which allow the relevant protease to digest the fusion protein of the invention and thereby liberate the recombinant peptide or protein of the invention therefrom. The liberated peptide or protein can then be isolated from the fusion partner by subsequent chromatographic separation.

[00224] Fusion partners may also include within their scope "epitope tags", which are usually short protein sequences for which a specific antibody is available.

[00225] As hereinbefore mentioned, peptide or proteins of the invention may be produced by culturing a host cell transformed with an expression construct comprising a nucleic acid encoding a peptide or protein of the invention. The conditions appropriate for expression will vary with the choice of expression vector and the host cell. For example, a nucleic acid sequence of the invention may be modified for successful or improved expression in a given host cell. Modifications include altering nucleotides depending on preferred codon usage of the host cell. Alternatively, or in addition, a nucleic acid sequence of the invention may be modified to accommodate host specific splice sites or lack thereof. These modifications may be ascertained by one skilled in the art.

[00226] Host cells for expression may be prokaryotic or eukaryotic.

[00227] Useful prokaryotic host cells are bacteria.

[00228] A typical bacteria host cell is a strain of E. coli.

[00229] Useful eukaryotic cells are yeast, SF9 cells that may be used with a baculovirus expression system, and other mammalian cells.

[00230] The recombinant peptide or protein of the invention may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook, et al., MOLECULAR CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989), incorporated herein by reference, in particular Sections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds., Ausubel et al. , (John Wiley & Sons, Inc. 1995-1999), incorporated herein by reference, in particular Chapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al., (John Wiley & Sons, Inc. 1995-1999) which is incorporated by reference herein, in particular Chapters 1, 5 and 6.

[00231] In one embodiment, nucleic acid homologs may encode peptide and protein homologs of the invention, inclusive of variants, fragments and derivatives thereof.

[00232] In yet another embodiment, nucleic acid homologs are nucleic acids having one or more codon sequences altered by taking advantage of codon sequence redundancy.

[00233] A particular example of this embodiment is optimization of a nucleic acid sequence according to codon usage by a selected organism as is well known in the art. This can effectively "tailor" a nucleic acid for optimal expression in a particular organism, or cells thereof, where preferential codon usage has been established.

[00234] In another embodiment, nucleic acid homologs may share at least 50% or 60%, preferably at least 62% or 70%, more preferably at least 80%, and even more preferably at least 90%, 95%, 98%, 99% or 100% sequence identity with the nucleic acids of the invention.

[00235] In yet another embodiment, nucleic acid homologs may hybridise to nucleic acids of the invention, including fragments, under at least low stringency conditions, preferably under at least medium stringency conditions and more preferably under high stringency conditions. [00236] "Hybridise and Hybridisation" is used herein to denote the pairing of at least partly complementary nucleotide sequences to produce a DNA-DNA, RNA-RNA or DNA-RNA hybrid. Hybrid sequences comprising complementary nucleotide sequences occur through base -pairing.

[00237] In DNA, complementary bases are:

(i) A and T; and

(ii) C and G.

[00238] In RNA, complementary bases are:

(i) A and U; and

(ii) C and G.

[00239] In RNA-DNA hybrids, complementary bases are:

(i) A and U;

(ii) A and T; and

(iii) G and C.

[00240] Modified purines (for example, inosine, methylinosine and methyladenosine) and modified pyrimidines (thiouridine and methylcytosine) may also engage in base pairing.

[00241] "Stringency" as used herein, refers to temperature and ionic strength conditions, and presence or absence of certain organic solvents and/or detergents during hybridisation. The higher the stringency, the higher will be the required level of complementarity between hybridizing nucleotide sequences.

[00242] "Stringent conditions" designates those conditions under which only nucleic acid having a high frequency of complementary bases will hybridize.

[00243] Reference herein to low stringency conditions includes and encompasses: -

(i) from at least about 1% v/v to at least about 15% v/v formamide and from at least about IM to at least about 2M salt for hybridisation at 42°C, and at least about IM to at least about 2M salt for washing at 42°C; and

(ii) 1% Bovine Serum Albumin (BSA), ImM EDTA, 0.5M NaHP0 ₄ (pH 7.2), 7% SDS for hybridization at 65°C, and (i) 2xSSC, 0.1% SDS; or (ii) 0.5% BSA, ImM EDTA, 40mM NaHP0 ₄ (pH 7.2), 5% SDS for washing at room temperature. [00244] Medium stringency conditions include and encompass: -

(i) from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for hybridisation at 42°C, and at least about 0.5M to at least about 0.9M salt for washing at 42°C; and

(ii) 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5M NaHP0 ₄ (pH 7.2), 7% SDS for hybridization at 65°C and (a) 2 x SSC, 0.1 % SDS; or (b) 0.5% BSA, ImM EDTA, 40mM NaHP0 ₄ (pH 7.2), 5% SDS for washing at 42°C.

[00245] High stringency conditions include and encompass: -

(i) from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01M to at least about 0.15M salt for hybridisation at 42°C, and at least about 0.01M to at least about 0.15 M salt for washing at 42°C;

(ii) 1 % BSA, 1 mM EDTA, 0.5 M NaHP0 ₄ (pH 7.2), 7% SDS for hybridization at 65°C, and (a) 0.1 x SSC, 0.1% SDS; or (b) 0.5% BSA, ImM EDTA, 40mM NaHP0 ₄ (pH 7.2), 1% SDS for washing at a temperature in excess of 65 °C for about one hour; and

(iii) 0.2 x SSC, 0.1% SDS for washing at or above 68°C for about 20 minutes.

[00246] In general, the T _m of a duplex DNA decreases by about 1 °C with every increase of 1 % in the number of mismatched bases.

[00247] Notwithstanding the above, stringent conditions are well known in the art, such as described in Chapters 2.9 and 2.10 of Ausubel et al., supra, which are herein incorporated by reference. A skilled addressee will also recognize that various factors can be manipulated to optimize the specificity of the hybridization. Optimization of the stringency of the final washes can serve to ensure a high degree of hybridization.

[00248] Typically, complementary nucleotide sequences are identified by blotting techniques that include a step whereby nucleotides are immobilized on a matrix (preferably a synthetic membrane such as nitrocellulose), a hybridization step, and a detection step. Southern blotting is used to identify a complementary DNA sequence; Northern blotting is used to identify a complementary RNA sequence. Dot blotting and slot blotting can be used to identify complementary DNA/DNA, DNA/RNA or RNA/RNA polynucleotide sequences. Such techniques are well known by those skilled in the art, and have been described in Ausubel et al., supra, at pages 2.9.1 through 2.9.20, herein incorporated by reference. [00249] According to such methods, Southern blotting involves separating DNA molecules according to size by gel electrophoresis, transferring the size-separated DNA to a synthetic membrane, and hybridizing the membrane bound DNA to a complementary nucleotide sequence.

[00250] In dot blotting and slot blotting, DNA samples are directly applied to a synthetic membrane prior to hybridization as above.

[00251] An alternative blotting step is used when identifying complementary nucleic acids in a cDNA or genomic DNA library, such as through the process of plaque or colony hybridisation. Other typical examples of this procedure are described in Chapters 8-12 of Sambrook et al., supra which are herein incorporated by reference.

[00252] Typically, the following general procedure can be used to determine hybridisation conditions. Nucleic acids are blotted/transferred to a synthetic membrane, as described above. A nucleic acid sequence of the invention is labeled as described above, and the ability of this labeled nucleic acid to hybridise with an immobilized nucleotide sequence analysed.

[00253] A skilled addressee will recognise that a number of factors influence hybridisation. The specific activity of radioactively labeled polynucleotide sequence should typically be greater than or equal to about 10 ⁸ d m g to provide a detectable signal. A radiolabelled nucleotide sequence of specific activity 10 ⁸ to 10 ⁹ d m g can detect approximately 0.5 μg of DNA. It is well known in the art that sufficient DNA must be immobilised on the membrane to permit detection. It is desirable to have excess immobilised DNA, usually 10 μg. Adding an inert polymer such as 10% (w/v) dextran sulphate (MW 500,000) or polyethylene glycol 6000 during hybridisation can also increase the sensitivity of hybridisation (see Ausubel et al, supra at 2.10.10).

[00254] To achieve meaningful results from hybridisation between a nucleic acid immobilised on a membrane and a labeled nucleic acid, a sufficient amount of the labeled nucleic acid must be hybridised to the immobilised nucleic acid following washing. Washing ensures that the labeled nucleic acid is hybridised only to the immobilised nucleic acid with a desired degree of complementarity to the labeled nucleic acid.

[00255] Methods for detecting labeled nucleic acids hybridised to an immobilised nucleic acid are well known to practitioners in the art. Such methods include autoradiography, chemiluminescent, fluorescent and colorimetric detection.

[00256] Nucleic acid homologs of the invention may be prepared according to the following procedure: (i) obtaining a nucleic acid extract from a suitable host, for example a bacterial species;

(ii) creating primers, which are optionally degenerate, wherein each comprises a portion of a nucleic acid sequence of the invention; and

(iii) using said primers to amplify, via nucleic acid amplification techniques, one or more amplification products from said nucleic acid extract.

[00257] As used herein, an "amplification product" refers to a nucleic acid product generated by nucleic acid amplification techniques.

[00258] Suitable nucleic acid amplification techniques are well known to the skilled addressee, and include PCR as for example described in Chapter 15 of Ausubel et al. supra, which is incorporated herein by reference; strand displacement amplification (SDA) as for example described in U.S. Patent No. 5,422,252 which is incorporated herein by reference; rolling circle replication (RCR) as for example described in Liu et al, 1996, J. Am. Chem. Soc. 118: 1587 and International application WO 92/01813; and Lizardi and Caplan, International Application WO 97/19193, which are incorporated herein by reference; nucleic acid sequence-based amplification (NASBA) as for example described by Sooknanan et al., 1994, Biotechniques 17: 1077, which is incorporated herein by reference; ligase chain reaction (LCR) as for example described in International Application WO89/09385 which is incorporated herein by reference; and Q-β replicase amplification as for example described by Tyagi et al., 1996, Proc. Natl. Acad. Sci. USA 93:5395 which is incorporated herein by reference. Preferably, amplification is by PCR using primers.

Antibodies

[00259] The invention also contemplates antibodies against the isolated peptides or proteins of the invention, including fragments, variants and derivatives thereof. Antibodies of the invention may be polyclonal or monoclonal. Well-known protocols applicable to antibody production, purification and use may be found, for example, in Chapter 2 of Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons NY, 1991-1994) and Harlow, E. & Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor Laboratory, 1988, which are both herein incorporated by reference.

[00260] The inventors have detected antibodies in the serum of animals with acute chlamydial infection in relation to peptides 1-4 (SEQ ID NOs: l-4). [00261] Generally, antibodies of the invention bind to or conjugate with a peptide, protein, fragment, variant, derivative, mimetic or pharmaceutically acceptable salt of the invention. For example, the antibodies may comprise polyclonal antibodies. Such antibodies may be prepared for example by injecting a peptide, protein, fragment, variant, derivative, mimetic or pharmaceutically acceptable salt of the invention into a production species, which may include mice or rabbits, to obtain polyclonal antisera. Methods of producing polyclonal antibodies are well known to those skilled in the art. Exemplary protocols which may be used are described for example in Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY, supra, and in Harlow & Lane, 1988, supra.

[00262] In lieu of the polyclonal antisera obtained in the production species, monoclonal antibodies may be produced using the standard method as for example, described in an article by Kohler & Milstein, 1975, Nature 256, 495, which is herein incorporated by reference, or by more recent modifications thereof as for example, described in Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY, supra by immortalizing spleen or other antibody producing cells derived from a production species which has been inoculated with one or more of the peptide, protein, fragment, variant, derivative, mimetic or pharmaceutically acceptable salt of the invention.

[00263] The invention also includes within its scope antibodies which comprise Fc or Fab fragments of the polyclonal or monoclonal antibodies referred to above. Alternatively, the antibodies may comprise single chain Fv antibodies (scFvs) against the peptide, protein, fragment, variant, derivative, mimetic or pharmaceutically acceptable salt of the invention. Such scFvs may be prepared, for example, in accordance with the methods described respectively in United States Patent No 5,091,513, European Patent No 239,400 or an article by Winter & Milstein, 1991, Nature 349 293, which are incorporated herein by reference.

[00264] The antibodies of the invention may be used for affinity chromatography in isolating natural or recombinant peptides or proteins of the invention. For example, reference may be made to immunoaffinity chromatographic procedures described in Chapter 9.5 of Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY, supra.

[00265] Antibodies may be purified from a suitable biological fluid of the animal by ammonium sulphate fractionation, affinity purification or by other methods well known in the art. Exemplary protocols for antibody purification are given in Sections 10.11 and 11.13 of Ausubel et al, supra, which are herein incorporated by reference.

[00266] Immunoreactivity of the antibody against the native or parent peptide or protein may be determined by any suitable procedure such as, for example, Western blot. Pharmaceutical compositions

[00267] A further feature of the invention is the use of peptides or proteins of the invention, including fragments, variants, derivatives, mimetics or pharmaceutically acceptable salts thereof and/or nucleic acids encoding the peptides or proteins, including fragments thereof, described herein as actives in a pharmaceutical composition. Preferably, the peptide or protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs: l-47; more preferably the peptide or protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs: l -12. Even more preferably, the peptide or protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs: l-4.

[00268] One aspect of the invention relates to a pharmaceutical composition comprising one or more peptides or proteins and/or nucleic acids of the invention as described herein, including fragments, variants, derivatives, mimetics or pharmaceutically acceptable salts thereof. In a preferred form, the pharmaceutical composition comprises a plurality of peptides, proteins and/or nucleic acids of the invention. It will be appreciated that a pharmaceutical composition comprising a plurality of antigens may provide an improved immune response against the pathogen, namely one or more forms of Chlamydia. Accordingly, a pharmaceutical composition preferably comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-five, thirty or more different peptides, proteins and/or nucleic acids of the invention.

[00269] The actives, in such composition, may be referred to as "immunogenic agents" which are capable of eliciting an immune response in a subject, including an animal or human. An immunogenic agent may be in the form of an immunotherapeutic composition, vaccine or antigen presenting cell loaded or pulsed with an antigen. The antigen presenting cell may be loaded or pulsed with antigen by contacting the cell with an antigen, for example a peptide or protein, fragment, variant, derivative, mimetic or pharmaceutically acceptable salt of the invention. The antigen presenting cell may be, for example, a dendritic cell.

[00270] A preferred form of a pharmaceutical composition is an immunotherapeutic composition. An immunotherapeutic composition preferably is a vaccine.

[00271] An immunotherapeutic composition may provide partial protection or partial improvement from a Chlamydia infection, and need not provide complete protection or treatment of Chlamydia infection to be useful. For example, partial protection or partial improvement from a Chlamydia infection may allow for a host immune system to overcome the infection, may improve health of the patient and/or may be combined with other treatments. In a preferred form, the immunotherapeutic composition or vaccine is capable of preventing onset of symptoms of disease caused by a Chlamydia infection. In another preferred form, the immunotherapeutic composition or vaccine is capable of treating symptoms of disease caused by a Chlamydia infection. Such diseases include: sexually transmitted disease, trachoma, LGV, arthritis, neonatal inclusion conjunctivitis, pneumonia and urethritis and cervicitis in women and the sequelae of these diseases include pelvic inflammatory disease, ectopic pregnancy, tubal factor infertility, epididymitis, proctitis and reactive arthritis. Other examples of such diseases include: polyarthritis and encephalomyelitis.

[00272] Preferably, the immunotherapeutic composition or vaccine is capable of interacting with Chlamydia during at least one stage of the organism's life cycle, for example during an intracellular stage and/or during an extracellular stage. For example, during an intracellular stage, a systemic cell-mediated immunity is preferred and during an extracellular stage a local mucosal IgA, and possibly IgG, response is preferred.

[00273] Suitably, the pharmaceutical composition comprises a pharmaceutically acceptable carrier or excipient. By "pharmaceutically-acceptable carrier or excipient" it is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of carriers, well known in the art may be used. These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulphate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water.

[00274] Any suitable route of administration may be employed for providing a subject with the pharmaceutical composition of the invention. For example, intranasal, transdermal, oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular and the like may be employed. Intranasal, transdermal, intra-muscular and subcutaneous application may be appropriate for administration of immunogenic agents of the present invention. Intranasal and transcutaneous administration in one preferred form includes use of cholera toxin and CpG- oligonucleotides as adjuvants. CpG-oligonucleotides are thought to induce primarily a Thl immune response and cholera toxin is thought to induce mucosal IgA when administered orally or intranasally, but induces an IgG response when administered transcutaneously, see for example Berry et al, 2004, Infect Immun 72: 1019, incorporated herein by reference. Skin penetration enhancers, such as chemical penetration enhancers including DMSO and electrically assisted methods including iontohoresis, may also be used as described for example in Barry, 2004, Nature Biology 22: 165.

[00275] Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of the therapeutic agent may be affected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, the controlled release may be affected by using other polymer matrices, liposomes and/or microspheres.

[00276] Pharmaceutical compositions of the present invention suitable for administration may be presented as discrete units such as vials, capsules, sachets or tablets each containing a predetermined amount of one or more immunogenic agent of the invention, as a powder, or granules, or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of a pharmacy but all methods include the step of bringing into association one or more immunogenic agents as described above with the carrier, which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the agents of the invention with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.

Vaccines

[00277] The above compositions may be therapeutic or prophylactic vaccines comprising one or more peptides, proteins and/or nucleic acids of the invention, or respective fragments, variants, derivatives, mimetics or pharmaceutically acceptable salts thereof. Preferably, the vaccine comprises a plurality of different peptides, proteins and/or nucleic acids as described herein. Preferably, the vaccine prevents or treats a Chlamydia infection. More preferably, the vaccine in one embodiment prevents or treats an animal infecting form of Chlamydia when administered to a subject animal. In another embodiment, the vaccine prevents or treats a human infecting form of Chlamydia when administered to a human patient. A pharmaceutical composition and vaccine need not completely prevent or treat a Chlamydia infection to be useful and reduced infection or reduced severity of infection is also considered a useful benefit and advantage. [00278] Accordingly, the invention extends to the production of vaccines comprising one or more actives (peptide, protein and/or nucleic acid) of the invention. Any suitable procedure is contemplated for producing such vaccines. Exemplary procedures include, for example, those described in NEW GENERATION VACCINES (1997, Levine et al, Marcel Dekker, Inc. New York, Basel Hong Kong) which is incorporated herein by reference.

[00279] An active according to the invention can be mixed, conjugated or fused with other antigens, including B or T cell epitopes of other antigens. In addition, it can be conjugated to a carrier as described below.

[00280] When a haptenic peptide or protein of the invention is used (i.e., a peptide or protein which reacts with cognate antibodies, but cannot itself elicit an immune response), it can be conjugated with an immunogenic carrier. Useful carriers are well known in the art and include for example: thyroglobulin; albumins such as human serum albumin; toxins, toxoids or any mutant cross reactive material (CRM) of the toxin from tetanus, diphtheria, pertussis, Pseudomonas, E. coli, Staphylococcus, and Streptococcus; polyamino acids such as poly(lysine: glutamic acid); influenza; Rotavirus VP6, Parvovirus VP1 and VP2; hepatitis B virus core protein; hepatitis B virus recombinant vaccine and the like. Alternatively, a fragment or epitope of a carrier protein or other immunogenic protein may be used. For example, a haptenic peptide or protein of the invention can be coupled to a T cell epitope of a bacterial toxin, toxoid or CRM. In this regard, reference may be made to U.S. Patent No 5,785,973 which is incorporated herein by reference.

[00281] The vaccines can also comprise a physiologically-acceptable carrier, diluent or excipient such as water, phosphate buffered saline and saline.

[00282] The vaccines and immunogenic compositions may include an adjuvant as is well known in the art. Any suitable adjuvant may be used, such as, e.g., aluminium hydroxide, aluminium hydroxyphosphate or any other suitable immunostimulating adjuvant. Suitable adjuvants include, but are not limited to adjuvants for use in animals or humans, for example, SBAS2, SBAS4, QS21 or ISCOMs.

[00283] The immunogenic agents of the invention may be expressed by attenuated viral hosts.

[00284] By "attenuated viral hosts" it is meant viral vectors that are either naturally, or have been rendered, substantially avirulent. A virus may be rendered substantially avirulent by any suitable physical (e.g., heat treatment) or chemical means (e.g., formaldehyde treatment). By "substantially avirulent" it is meant a virus whose infectivity has been destroyed. Ideally, the infectivity of the virus is destroyed without affecting the proteins that carry the immunogenicity of the virus. From the foregoing, it will be appreciated that attenuated viral hosts may comprise live viruses or inactivated viruses.

[00285] Attenuated viral hosts which may be useful in a vaccine according to the invention may comprise viral vectors inclusive of adenovirus, cytomegalovirus and preferably pox viruses such as vaccinia (see, e.g., Paoletti and Panicali, U.S. Patent No. 4,603,112 which is incorporated herein by reference) and attenuated Salmonella strains (see for example Stacker, U.S. Patent No. 4,550,081 which is herein incorporated by reference). Live vaccines are particularly advantageous because they lead to a prolonged stimulus that can confer substantially long-lasting immunity.

[00286] Multivalent vaccines can be prepared from one or more different epitopes of one or more peptides or proteins as described herein. An example of a preferred multivalent vaccine includes a nucleic acid encoding one or more epitopes of one or more Chlamydia peptides or proteins, including fragments, variants, derivatives, mimetics or pharmaceutically acceptable salts thereof. A preferred form of a nucleic acid suitable for use as a multivalent vaccine comprises a nucleic acid sequence comprising two or more of the peptide or protein fragments expressed as a fusion protein with no (i.e., contiguous) or minimal amino acids between adjacent peptide or protein fragments. For example, peptide 1 (SEQ ID NO: l) may be expressed as a fusion protein with any one of peptides 2-4 (SEQ ID NOs:2-4). The multivalent vaccine may encode 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or more peptide or protein antigens, including fragments or variants thereof. The multivalent vaccine may comprise one or more same peptide or protein antigens, for example, 1 , 2, 3, 4, 5, 6 or more of the same peptide or protein antigens including various fragments and variants thereof. The multivalent protein may also comprise B-cell and T-cell epitopes.

[00287] A recombinant vaccinia virus may be prepared to express a nucleic acid according to the invention. Upon introduction into a host, the recombinant vaccinia virus expresses the immunogenic agent, and thereby elicits a host response. For example, reference may be made to U.S. Patent No 4,722,848, incorporated herein by reference, which describes vaccinia vectors and methods useful in immunization protocols.

[00288] A wide variety of other vectors useful for therapeutic administration or immunization with the immunogenic agents of the invention will be apparent to those skilled in the art from the present disclosure. [00289] The nucleic acids of the invention may be used as a vaccine in the form of a "naked DNA" vaccine as is known in the art. For example, an expression vector of the invention may be introduced into a mammal, where it causes production of a peptide or protein in vivo, against which the host mounts an immune response as for example described in Barry, M. et al., (1995, Nature, 377:632-635) which is hereby incorporated herein by reference.

[00290] In one embodiment, the invention relates to a nucleic acid vaccine. It will be appreciated that the method of isolating the nucleic acids of the invention may involve steps of expressing a nucleic acid in an animal and assessing an immune response. Accordingly, the nucleic acids of the invention may be particularly useful for a nucleic acid vaccine.

Preparation of immunoreactive fragments

[00291] The invention also extends to a method of identifying an immunoreactive fragment of a peptide, protein, variant or derivatives according to the invention. This method essentially comprises generating a fragment of the peptide, protein, variant or derivative, administering the fragment to a mammal; and detecting an immune response in the mammal. The method for identifying the isolated nucleic acids and fragments includes expression in a host and detection of an immune response. Accordingly, the peptides, proteins and nucleic acids encoding the peptides or proteins may be useful in preparing immunological reagents such as antibodies.

[00292] Prior to testing a particular fragment for immunoreactivity in the above method, a variety of predictive methods may be used to deduce whether a particular fragment can be used to obtain an antibody that cross-reacts with the native antigen. These predictive methods may be based on amino-terminal or carboxy-terminal sequence as for example described in Chapter 11.14 of Ausubel et al., supra. Alternatively, or in addition, these predictive methods may be based on predictions of hydrophilicity as for example described by Kyte & Doolittle 1982, J. Mol. Biol. 157:105 and Hopp & Woods, 1983, Mol. Immunol. 20:483) which are incorporated by reference herein, or predictions of secondary structure as for example described by Choo & Fasman,1978, Ann. Rev. Biochem. 47:251), which is incorporated herein by reference.

[00293] Generally, a peptide or protein fragment consisting of 10 to 15 residues provides optimal results. Peptides or proteins fragments as small as 6 or as large as 20 residues have worked successfully. Such peptide or protein fragments may then be chemically coupled to a carrier molecule such as keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA) as for example described in Sections 11.14 and 11.15 of Ausubel et al., supra. [00294] The peptides or proteins fragments may be used to immunize an animal as for example discussed above. Antibody titres against the native or parent peptide or protein from which the peptide or protein fragment was selected may then be determined by, for example, radioimmunoassay or ELISA as for instance described in Sections 11.16 and 11.14 of Ausubel et al, supra.

Detection methods and kits

[00295] The present invention also provides a method and kit for detecting Chlamydia and/or one or more Chlamydia antigens in a biological sample. A "biological sample" refers to a sample derived or obtained from a biological source, for example a cell, tissue, organ, organism, including Chlamydia and components thereof, lysate, homogenate, biological fluid including mucus, blood, serum, plasma, urine or cerebrospinal fluid. A kit will comprise one or more agents described above depending upon the nature of the test method employed.

[00296] In this regard, the kits may include one or more of a peptide, protein, fragment, variant, derivative, mimetic, pharmaceutically acceptable salt, antibody, antibody fragment, nucleic acid or nucleic acid fragment according to the invention. The kits may also optionally include appropriate reagents for detection of labels, positive and negative controls, washing solutions, dilution buffers and the like.

[00297] A peptide, protein or antibody based detection kit may include (i) a peptide, protein or variant, derivative, mimetic, pharmaceutically acceptable salt thereof according to the invention (which may be used as a positive control), (ii) an antibody according to the invention (preferably a monoclonal antibody) which binds to a Chlamydia antigen or fragment thereof in (i), and (iii) a suitable means for detecting a complex formed between a target (e.g., a Chlamydia antigen in a sample) and the antibody in (ii), the detection means may include, e.g., colloidal gold, protein, enzyme, colour reagent and the like.

[00298] A peptide, protein or antibody in one form is capable of binding to a target protein common to one or more biovar, serovar, amino type, strain, variant or species of Chlamydia. Such a peptide, protein or antibody may be useful for identifying a range of different biovars, serovars, genotypes, strains, variants or species of Chlamydia in a biological sample. Alternatively, or in addition, a peptide, protein or antibody may be specific for a particular biovar, for example a trachoma biovar or lymphogranuloma venereum biovar; or specific for a particular serovar, e.g., serovars A, B, Ba, C, D, Da, E, F, G, H, I, la, J, K, LI, L2 or L3 of C. trachomatis, or a particular genotype, e.g., genotypes A, F, H and H of C. precorum. The binding of the peptide, protein or antibody with a target protein may be detected using methods common to the art, including biotin- avadin, biotin-streptavadin, enzymatic detection methods common for ELISA based detection methods, colour regents, fluorescent dyes, radioactive labels such ³² P or ³⁵ S, and metals, e.g., gold, Western blotting, dot blotting, affinity columns and the like.

[00299] It will be appreciated that peptide -protein or protein-protein interactions may be used to detect a target protein, e.g., a peptide or protein that may form a complex with a target protein, which may be detected. Also, substrates for a target protein or fragment thereof may be used to detect the presence of the target protein or fragment thereof. For example, a substrate for DNA gyrase subunit B, sulfite synthesis/biphosphate phosphatase, methionyl-tRNA synthetase, DNA helicase (urvD) and/or ATP synthase subunit I. It is also contemplated that a peptide, protein or antibody based kit in one form detects antibodies in a biological sample from a subject that bind to one or more Chlamydia proteins. Such antibodies may be detected following infection by Chlamydia.

[00300] A detection kit may be nucleic acid based. Such a kit will contain one or more particular agents, e.g., nucleic acids, peptides and proteins as described above, depending upon the nature of the test method employed. In this regard, the kits may include one or more nucleic acid encoding Chlamydia antigen(s), or fragments thereof, as described herein, inclusive of primers and probes according to the invention. Accordingly, the kits may include reagents for detection of labels, positive and negative controls, washing solutions, dilution buffers and the like. For example, a nucleic acid amplification based kit may include primers capable of hybridising with target nucleic acids encoding one or more Chlamydia antigens isolated from a biological sample. Such a biological sample in one embodiment may be isolated from an individual subject, preferably a human patient or animal patient. The primers may be capable of hybridising to a nucleic acid comprising a nucleotide sequence shared by one or more biovar, serovar, strain, genotype, variant or species of Chlamydia. Such primers may be useful for identifying a range of different biovars, serovars, genotypes, strains, variants or species of Chlamydia in a biological sample. Alternatively, or in addition, a primer may be specific for a particular biovar, e.g., a trachoma biovar or lymphogranuloma venereum biovar; or specific for a particular serovar, e.g., serovar A, B, Ba, C, D, Da, E, F, G, H, I, la, J, K, LI , L2 or L3; or specific for a particular genotype, e.g., genotype A, F, G or H. The nucleic acid primer may comprise any suitable number of contiguous nucleic acids, preferably between 5 and 200 nucleotides, e.g., 5, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175 or 200. The nucleic acid may be amplified according to methods known in the art, for example PCR. The amplified nucleic acid, or non-amplified nucleic acids, can be compared with reference nucleic acids using methods including for example, separation by molecular weight by gel electrophoresis, hybridisation methods including Northern and Southern blotting, microarrays and other methods common in the art. Non-amplification methods of detection may include isolation of mRNA from a sample and performing Northern blot analysis using a nucleic acid probe derived from one or more of the Chlamydia nucleic acids, including fragments, described herein. The design of the nucleic acid probe may be based on sequences that are common between Chlamydia biovars, serovars, genotypes, strains, variants or species, or sequences that share some sequence identity with selected Chlamydia biovars, serovars, genotypes, strains, variants or species, or sequences that share sequence identity with all known Chlamydia biovars, serovars, genotypes, strains, variants or species. A nucleic acid may be detected using labels common in the art including, e.g., fluorescent dyes, radioactive labels such ³² P or ³⁵ S, enzymes and metals, including gold.

Microarrays

[00301] A microarray also uses hybridization-based technology that, for example, may allow detection and/or isolation of a nucleic acid by way of hybridization of complementary nucleic acids. A microarray provides a method of high throughput screening for a nucleic acid in a sample that may be tested against several nucleic acids attached to a surface of a matrix or chip. In this regard, a skilled person is referred to Chapter 22 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Eds. Ausubel et al. John Wiley & Sons NY, 2000).

[00302] Each nucleic acid occupies a known location on an array. A nucleic acid target sample probe is hybridised with the array of nucleic acids and an amount or relative abundance of target nucleic acid hybridised to each probe in the array is determined. High-density arrays are useful for monitoring gene expression and presence of allelic markers which may be associated with disease. Fabrication and use of high density arrays in monitoring gene expression have been previously described, for example in WO 97/10365, WO 92/10588 and US Patent No. 5,677,195, all incorporated herein by reference. In some embodiments, high-density oligonucleotide arrays are synthesised using methods such as the Very Large Scale Immobilised Polymer Synthesis (VLSIPS) described in US Patent No. 5,445,934, incorporated herein by reference.

Expression library immunisation

[00303] Expression library immunisation (ELI) is an approach to vaccine production that has the potential to identify vaccine antigens. ELI has been described in US Patent 5,703,057 (Johnston et al) and AU Patent 764256 (CSIRO) for non-Chlamydia pathogens, however, similar methods taught by these patents may be used herewith. ELI is an attractive method to investigate vaccine antigens because unlike some more traditional vaccine strategies, it requires no prior knowledge of antigenic targets and has the potential to screen every gene in the host pathogen genome. The process of screening the genome of a pathogen through a disease model also has the ability to induce both cellular and humoral immune responses so that no assumptions regarding the best immune response need to be made.

[00304] ELI involves the construction of a genomic library by fragmenting the pathogen DNA (or cDNA) and incorporating these library fragments into eukaryotic expression vectors. Pools, or sub libraries, of these clones then are screened through a disease model to demonstrate whether the sub library can confer a degree of protection against a challenge infection by the pathogen. The sub library or libraries that offer protection can then be sub fractionated to reduce the complexity of the genomic library and eventually identify protective clones (Johnston and Barry 1997), incorporated herein by reference.

[00305] ELI was first reported by Barry et al. (Barry et al. 1995) who demonstrated that DNA vaccination with genomic libraries of Mycoplasma pulmonis constructed in a eukaryotic expression vector could protect mice against infection. Since then there have been several reports of the use of this method in parasites (Johnston and Barry 1997; Brayton et al. 1998; Melby et al. 2000; Smooker et al. 2000). This work with ELI has used a variety of vectors for library construction; however, a major problem with all the libraries has been the large percentage of unproductive plasmids. Fragments of the pathogen genome that are in the wrong orientation, wrong reading frame or in intergenic regions will not express fusion proteins and hence are not able to induce an immune response. Recently, however, Moore et al. 2001 have developed improved vectors that allow screening for clones that are expressing a recombinant protein to overcome these problems. This was achieved by using a polyHis fusion partner and screening the transformed library in E.coli using polyHis detection methods. Unproductive clones, or those not producing a fusion protein were negative in the screen because small, unfused polyHis proteins have a very short half-life within the cells and any residual protein binds poorly to the membranes used in the screening process. In this manner the clones capable of expressing protein and hence those capable of inducing an immune response were isolated. The authors used these improved vectors for ELI against Mycoplasma hyopneumoniae in pigs to reduce a library of 20,000 clones to a protective group of just 96 clones. ELI is readily applicable to pathogens with small genomes such as Chlamydia because the genetic complement of such organisms can be represented in small libraries.

[00306] In the present specification and claims, the word 'comprising' and its derivatives including 'comprises' and 'comprise' include each of the stated integers but does not exclude the inclusion of one or more further integers.

[00307] Reference throughout this specification to One embodiment' or 'an embodiment' means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearance of the phrases 'in one embodiment' or 'in an embodiment' in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more combinations.

[00308] In compliance with the statute, the invention has been described in language more or less specific to structural or methodical features. It is to be understood that the invention is not limited to specific features shown or described since the means herein described comprises preferred forms of putting the invention into effect. The invention is, therefore, claimed in any of its forms or modifications within the proper scope of the appended claims (if any) appropriately interpreted by those skilled in the art.

EXAMPLES

EXAMPLE 1

Experimental Procedures

Animals

[00309] Animals were selected from a population of koalas inhabiting the Moreton Bay Region (MBR) of South-East Queensland, Australia. The animals selected were sexually mature adult koalas (> 1 year old; males and females). The animals were selected on the basis of presenting clinically healthy, but may have been asymptomatically Chlamydia infected.

[00310] A total of 20 koalas were monitored for a six month period. The koalas were divided into four cohorts (each consisting of five koalas) on the basis of C. precorum infection load by qPCR at the initial stage of selection. The groups were: (1) non-vaccinated, Chlamydia positive koalas (Bubbles, Cougar, Karen, Coco and Mango); (2) vaccinated, Chlamydia positive koalas (Tash, Bev, Old Bean, Fiona and Poppy); (3) non-vaccinated, Chlamydia negative koalas (Kev, Teena, Phil, Red Queen and Gauthier); and (4) vaccinated, Chlamydia positive koalas (Robyn, Pepper, Maya, Hunky Harry and Randall).

[00311] Antibody characteristics were analysed for all 20 koalas at their initial capture. Additionally, 10 of the animals received an rMOMP-based vaccine (discussed below) and their post-vaccination responses were monitored.

[00312] Another four non-vaccinated koalas were included in the antibody characteristics analysis to measure the impact of infection load over time with antibody titres.

[00313] Plasma samples were collected from all animals at 0, 2 and 6 month time points.

Vaccine

[00314] Animals in the vaccinated groups received three doses of an rMOMP based vaccine via a subcutaneous route as previously characterised in Waugh, C.A., et al. (2015) Vaccine in press, incorporated herein by reference.

[00315] E. coli strain pLysS competent cells were used for molecular cloning, protein expression and purification of the rMOMP protein. The cells were expressed in LB broth at 37°C with constant shaking and purified as per Kollipara, A. et al. 2012, incorporated herein by reference.

[00316] The purified rMOMP protein was then used for vaccination and ELISA assays.

[00317] Once vaccinated, the animals were released back into their natural habitat and tracked with a wildlife telemetry system (K-Tracker, LX Solutions Pty Ltd). The vaccinated animals were re-captured at 1 month intervals to receive their second and third doses of the vaccine.

Sample collection

[00318] Selected animals were captured at 0, 2 and 6 month time points for collecting samples and a full health assessment by Endeavour Veterinary Ecology Pty Ltd. A 1ml blood sample was collected from each animal from the cephalic vein while the animal was anaesthetised. Samples were collected in a vacutainer tube (containing EDTA - Ethylinediaminetetraacetic acid as anticoagulant; Interpath Services). The tubes were then labelled with the animal's name and date, stored at 4°C away from direct sunlight and processed within 5 hours of collection to separate the plasma via centrifugation. The plasma was then kept at -80°C for further testing. Swab samples were also taken from the ocular and urogenital (UGT) sites of the animals and stored immediately at -20°C until analysis.

Screening of C. precorum infections [00319] The ocular and UGT samples were screened for the presence of C. pecorum infection by a species-specific 16S rRNA quantitative PCR as per the procedures described in Marsh, J. et al. BMC microbiology (2011) 11:77, incorporated herein by reference.

Koala C. precorum-specific IgG ELISAs

[00320] Enzyme linked immunosorbent assays were performed using either (a) rMOMP or (b) UV inactivated whole EBs (elementary bodies) on the plasma samples at the 0, 2 and 6 month time points as per Kollipara A. et al. Vaccine 2012, 30:1875-1885, incorporated herein by reference.

C. precorum in vitro neutralising assay

[00321] In vitro neutralisation assays were performed on animal plasma samples collected at 0, 2 and 6 month time points according to Kollipara A. et al. Vaccine 2012, 30:1875-1885, incorporated herein by reference. All plasma samples were diluted at 1 : 10 prior to assaying. The assays were also completed with the plasma after successfully removing the targeted peptides. The background neutralisation was determined by using animal plasma that had no infection. The actual neutralization was then determined by subtracting this background from each individual to get the final neutralisation. The results were expressed as fold change neutralisation.

MOMP epitope mapping by Pepscan ELISAs

[00322] The biotinylated peptide ELISA was performed for plasma samples as per Kollipara, A. et al. Plos one (2013) 8:e748080, incorporated herein by reference to identify the specific MOMP peptides recognised by vaccinated animals and C. precorum positive animals.

[00323] 88 15-mer peptides were designed (overlapping by 9 amino acids) that spanned the full length of koala C. precorum H strain MOMP protein (see SEQ ID NO:51).

[00324] A 96 well streptavidin-coated plate pre -blocked with BSA (Thermo, Fischer Scientific, Melbourne, Australia) was then coated with the individual peptides at a concentration of 2μg/well in IX Phosphate Buffered Saline Tween-20 (PBS-T) and incubated at room temperature (22°C) for 2 hrs.

[00325] Post incubation, the wells were washed 3X with PBS-T and the individual plasma samples (diluted 1/1000) were added to the wells (100 μΐ/well), respectively. The plates were incubated overnight at 4°C followed by washing 4X with PBS-T.

[00326] The plate were then incubated with secondary and tertiary anitbodues with sheep anti- koala IgG (1 :4000 dilution) and HRP labelled rabbit anti-sheep IgG (1 : 1000 dilution), respectively. After 1 hr incubation, the plate was then washed with PBS (Phosphate buffer solution) and ABTS [2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid), Southern Biotech, Alabama, USA] solution was added and incubated for 10 mins to observe greenish colour development. The optical density was measured at 405nm wavelength (Bio-Rad,North Ryde, Australia). The background for the each plasma sample was calculated from the mean plus twice the standard deviation of the negative wells (no sera added). A positive response was regarded as an optical absorbance value of <0.50.

[00327] In subsequent experiments, the positive peptides wered utilised to coat the streptavidin plate at a concentration of 2μg/well and performed the standard ELISA as described.

Characterisation of specific anti-epitope antibodies responsible for invitro neutralisation

[00328] Once epitope specificity for sera from post-vaccinated animals had been identified, further experiments were undertaken to determine if the in vitro neutralisation capacity of the sera was explained by the antibodies to these epitopes. A streptavidin plate was coated with specific peptides (4, 28, 41, and 42) at a concentration of 2μg/well and incubated at room temperature for 2 hrs.

[00329] Post-incubation, the plates were washed with 3x PBS-T and incubated with the positive plasma samples at 4° C overnight. This step allows the plasma to absorb out the 4 specific peptides. Post-incubation plasma was then collected and the biotynalated peptide ELISA as described above was again conducted.

[00330] C. pecorum specific neutralising antibodies for the pre- and post absorption plasma were measured. The pre-immunized plasma samples were used as background neutralisation to determine the actual neutralisation.

Statistics

[00331] Statistical analyses were performed using Graph-Pad Prism version 6 (Graph Pad Software, LaJolla, CA, USA). Correlation analysis was performed by spearman rank correlation test using Software STATA/IC-11 (StataCorp, 4905, LakewayDrive, College station, TX 77845, USA). The P value for significance was set at < 0.05.

RESULTS

Antibody responses in naturally infected animals and in animals post vaccination [00332] The animal groups (prior to any vaccination) can be divided into C. pecorum PCR negative and PCR positive sub-groups. All five C. pecorum PCR negative animals (non- vaccinated) did not have any antibodies to either C. pecorum rMOMP protein or to whole chlamydial EBs (see Figure 1; A and B, respectively).

[00333] By comparison, all five (non-vaccinated) animals, which were C. pecorum PCR positive at the time of sampling, had antibodies that recognised both rMOMP protein (titres ranging from 0.5xl0 ⁶ to 2xl0 ⁶ EPT) as well as whole chlamydial EBs (titres ranging from 0.5xl0 ³ to lxlO ³ EPT; see Figure 1). Given that it was not know when each animal was initially infected, it was not surprising that the titres remained relatively constant across the 6 month study period.

[00334] Following vaccination, all ten animals that were vaccinated produced antibodies to both rMOMP protein as well as whole chlamydial EBs (see Figure 2). Interestingly, animals that were PCR positive at the time of vaccination produced stronger antibody responses than those that were PCR negative (although there was animal to animal variation). This was particularly evident with the anti-rMOMP responses, where 4 out of 5 PCR positive animals produced titres of around 3- 4xl0 ⁶ (see Fi gure 2C) _, compared to peak titres for the C. pecorum PCR negative vaccinated animals of around lxlO ⁶ , with a single animal at 2xl0 ⁶ (see Figure 2A). These differences were not observed for the antibody level measured against whole chlamydial EBs.

In vitro neutralisation levels were boosted significantly following vaccination

[00335] Naturally infected animals (C. pecorum PCR positive at the time of analysis) did have in vitro neutralisation titres, although these levels were low, ranging from 0.5 to 2.0 fold change with a mean of 1.0 fold change (see Figure 3C). When these PCR positive animals received the vaccine, their in vitro neutralisation levels were boosted significantly, increasing to levels of 2.5 to 8.0 fold change, with a mean of 4.8 fold change (see Figure 3D). Animals in the vaccinated C. pecorum PCR negative group also developed in vitro neutralising antibodies (1.5 to 3.0 fold change with a mean of 2.7 fold change; see Figure 3B), although their levels were not as high as for C. pecorum PCR positive animals.

Lower plasma antibodies titres correlate with higher infectious burdens in naturally infected animals

[00336] In the naturally infected groups (C. pecorum PCR positive at the time of analysis, which was increased to a total of 14 animals for this aspect), the chlamydial infection level of the animal (as measured by quantitative PCR at the UGT site) was compared with the plasma antibody titres (measured against rMOMP protein). A strong and statistically significant correlation (p < 0.01) was found between higher infection load and lower antibody titre (see Figure 4).

C. precorum PCR positive animals, as well as vaccinated animals, show different unique peptide antibody response profiles

[00337] The pepscan methodology was used to examine epitope specificity of the antibodies produced (a) in diseased animals compared to non-diseased (but C. pecorum PCR positive animals), and (b) in C. pecorum PCR negative animals post vaccination. It was found that diseased animals had plasma antibodies that specifically recognised a unique set of rMOMP epitopes (5, 33, 79 and 85) that were not recognised by plasma antibodies from healthy koalas (see Figure 5).

[00338] By comparison, vaccinated animals produced antibodies that recognised a different unique set of epitopes (80% to 90% of animals tested had responses to epitopes 4, 28 and 41 and 40% to 60% of animals tested had a response to epitope 42).

In vitro neutralisation of post-vaccination sera is associated with antibodies against specific MOMP epitopes

[00339] It was observed that the in vitro neutralisation titres were highest in animals that were previously infected and then vaccinated (see Figure 3). To determine whether it was the antibodies to specific epitopes that were responsible for this in vitro neutralisation effect, peptides corresponding to epitopes 4, 28, 41 and 42 were used to absorb against plasma from infected and/or non-infected plus vaccinated animals. The plasma was then tested to confirm whether antibodies specific for the epitopes had been removed (see Figure 6). The plasma was then tested in an in vitro neutralisation assay (see Figure 6).

[00340] As can be seen from Figure 6, antibodies against epitopes 4, 28, 41 and 42 were successfully removed from the plasma. Compared to the pre-absorption plasma which had an in vitro neutralisation fold effect of 4.8 times, the post-absorption plasma had virtually no in vitro neutralising ability (fold change of 1.0). This confirms that all, or at least the majority, of the in vitro neutralisation was due to antibodies against these specific epitopes (4, 28, 41 and 42).

DISCUSSION

[00341] Chlamydial disease continues to be a major threat to the long term survival of koalas. Many individual or "isolated " near-urban populations are close to becoming extinct every year and while habitat destruction, vehicle injury and wild dogs are important issues, losses due to chlamydial infection and disease are a major factor (Polkinghorne, A., et al. 2013).

[00342] For this reason, the development of an effective chlamydial vaccine is critical.

[00343] Vaccine research in the mouse model in particular, has shown that an interferon-gamma T cell response is important (Brunham, R.C., Rey-Ladino, J., 2005), but also suggests that neutralizing antibodies have a role in protection (Rank, R.G., Whittum-Hudson, J.A., 2010).

[00344] From the present experiments, a number of new and highly relevant observations have been made that are not only relevant to chlamydial infections in koalas, but also show how the "C. pecorum-koala model" might be able to inform human C. trachomatis and animal C. precorum vaccine development.

[00345] A unique aspect from the present experiments was that C. pecorum positive animals as well as C. pecorum negative animals were vaccinated. It is not possible to do this in humans, as protocol requires that any Chlamydia positive individual be immediately treated with antibiotics. Perhaps not surprisingly, in the current experiments, higher antibody responses were found in vaccinated animals which had a current chlamydial infection. A different antibody kinetic response was also observed, reminiscent of a secondary response in these animals. This antibody response, to a linear protein in the form of rMOMP vaccine, was also able to recognise conformational MOMP on whole EBs. This is critical and very promising for successful vaccine development.

[00346] The functionality of these antibodies was then examined by assaying their ability to neutralize C. pecorum infections in an in vitro neutralisation assay. The in vitro neutralisation ability of antibodies from naturally infected animals were first examined and found to have very low, to nil, antibodies capable of neutralisation. This suggests that the antibody response to whole live chlamydiae in a natural infection somehow results in antibodies of specificity that do not negate the infection. By comparison, plasma from vaccinated animals (both animals with a current infection but also C. precorum negative animals) does neutralise C. pecorum infections in vitro. Somewhat unexpectedly, however, the plasma from animals which were previously naturally infected (and not protected) but then vaccinated, produced a significantly higher in vitro neutralisation effect. This suggests that previous chlamydial infection boosted the humoral response with the administration of the rMOMP vaccine.

[00347] Given the promising antibody response following vaccination, the pepscan methodology was used to characterise the type of antibodies being produced. Two unique epitope profiles were identified. One profile was found in naturally infected animals that progressed to disease, i.e., not protected. By comparison, vaccinated animals produced a different, unique profile, against 3-5 key epitopes. To confirm the specificity of these key epitopes identified, individual peptides for the key epitopes were used to absorb the specific antibodies out of these groups, and the in vitro neutralising ability for the plasma was then re-tested. This showed a marked drop from a strong in vitro neutralisation ability to virtually zero.

[00348] Different epitopes are recognized in animals with a natural chlamydial infection compared to vaccinated koalas (both naturally infected and non-infected animals). From a vaccine development perspective this is important as the 3-4 key epitopes (induced by the vaccine) were in the conserved domains of C. pecorum (Kollipara, A., et al. 2013). It is posited that these conserved epitopes may be responsible for a wider range of cross protection.

EXAMPLE 2 -PEPTIDE VACCINES

Experimental Procedures

Animals

[00349] Animals were selected from a population of koalas from Lone Pine Koala Sanctuary, Fig Tree Pocket, Queensland, Australia. The animals selected were sexually mature male adult koalas (> 1 year old). The animals selected were clinically healthy and free of any chlamydia infections.

[00350] A total of 10 male koalas were selected. The 10 koalas were divided into two groups, namely: Group 1 consisting of five koalas which received a rMOMP based vaccine (discussed below); and Group II consisting of the remaining five koalas (Jervis, Ficus, Sargen, Aster and Perkings) which received a vaccine including two peptides.

Vaccine

[00351] Animals in Group I received a single dose of an rMOMP based vaccine via a subcutaneous route as previously characterised in Khan, S.A., et al. Vaccine 2014 7;32(44):5781-6, incorporated herein by reference. The rMOMP based vaccine was mixed with a three -component adjuvant as also previously characterised in Khan, S.A., et al. Vaccine 2014 7;32(44):5781-6.

[00352] Animals in Group II received a single dose of a vaccine composition including 50 μg of peptide 1 corresponding to epitope 4 of MOMP from C. precorum genotypes A, F and H (SEQ ID NO: 1) and 50 μg of peptide 3 corresponding to epitopes 41/42/43 of from C. precorum genotypes A, F and H (SEQ ID NO: 3). As with the vaccine administered to Group I, the vaccine composition was mixed with the three-component adjuvant as previously characterised in Khan, S.A., et al. Vaccine 2014 7;32(44):5781-6 prior to subcutaneous administration.

[00353] Once vaccinated, the animals were released back into their enclosures at Lone Pine Koala Sanctuary.

Sample Collection

[00354] Samples were collected from the animals at 6 and 12 week time points post- vaccination. The samples collected included whole blood samples - for plasma and lymphocyte analyses as well as swabs from ocular and penile sites.

Enzyme linked immunosorbent assay (ELISA)

[00355] Enzyme linked immunosorbent assays (ELISAs) were performed using either (a) rMOMP or (b) peptides 1 and 3 on the plasma samples at the 6 and 12 week time points as per Kollipara A. et al. Vaccine 2012, 30:1875-1885, incorporated herein by reference.

Measurement of anti-MOMP/anti-Peptide 1 and 3 lymphocyte proliferation response

[00356] Lymphocyte proliferation responses were assayed as per Khan, S.A., et al. Vaccine 2014 7;32(44):5781-6. Peripheral blood mononuclear cells (PBMCs) were collected from the whole blood samples collected at the 6 and 12 week time points. PBMCs were isolated on Ficoll gradients, labelled with carboxyfluorescein succinimidylester (CFSE) then stimulated with rMOMP or peptides 1 and 3 as a positive stimulator, and no antigen as a negative control. Proliferation of PBMCs was determined using a Beckman Coulter flow cytometer (FC500, GladesviUe, NSW, Australia).

RESULTS

[00357] Both the rMOMP based vaccine and the peptide -based vaccine were safe and none of the animals had any adverse reactions to the vaccines, either at the time of administration or in the weeks afterwards.

Antibody Responses at week 6

[00358] All Group II animals vaccinated with peptides 1 and 3 responded by making antibodies (detected in a peptide-specific ELISA) in their plasma at 6 weeks post-vaccination to epitopes 4 and 42 and to a lesser extent epitope 43 (see Table 2). Antibody Responses at week 12

[00359] All Group II animals vaccinated with the peptides produced a good antibody response to epitope 4. The response to this peptide was the strongest antibody response in all animals, with some animals (e.g., Ficus) producing very high levels of antibodies to this epitope (see Table 2).

[00360] Epitope 42 was in peptide 3 used for vaccination and while there was a response to this peptide in some of the animals (3 out of 5), the response (level of antibodies) was lower than that seen for epitope 4 (see Table 2).

[00361] Table 2— Group II Antibody Responses at weeks 6 and 12 (week 12 responses underlined).

DISCUSSION

[00362] Previous work has shown that koalas vaccinated with a 3-dose ISC adjuvant combined with a full length recombinant MOMP protein produces an antibody and cell mediated immune response. When examined, the specificity of this immune response (antibody response) was directed to specific epitopes along the MOMP protein, and these responses were different to those found in koalas which had a natural infection (which they could not control / had not mounted an effective immune response to). The inventor therefore designed a series of short peptides from the full length MOMP protein. The design of these peptides involved the selection of epitopes that reacted in previous MOMP trials, combined with knowledge of amino acid sequences that could contribute both B cell and T cell epitopes. The peptides were further refined as a result of the chemical synthesis process. Of the peptides designed, two were tested in this vaccine trial.

[00363] Even though the peptides were relatively short and even though they were not conjugated to a carrier (to enhance their immunogenicity), by 12 weeks post-vaccination all five Group II animals vaccinated with these two peptides had produced peptide-specific IgG antibodies in their plasma.

EXAMPLE 3— FURTHER PEPTIDE VACCINES Experimental Procedures

Animals

[00364] Animals will be selected from a population of koalas from Lone Pine Koala Sanctuary, Fig Tree Pocket, Queensland, Australia. The animals selected will be sexually mature male adult koalas (> 1 year old). The animals selected will be clinically healthy and free of any chlamydia infections.

[00365] A total of five or more male koalas may be selected. The five or more koalas receive a vaccine including two or more peptides.

Vaccine

[00366] Animals will receive a single dose of a vaccine composition including at least 50 μg of peptide 2 corresponding to epitope 28 of MOMP from C. precorum genotypes A, F and H (SEQ ID NO: 2) and at least 50 μg of peptide 4 corresponding to epitope 44 of from C. precorum genotypes A, F and H (SEQ ID NO: 4). The vaccine composition will be mixed with the three-component adjuvant as previously characterised in Khan, S.A., et al. Vaccine 2014 7; 32(44): 5781-6 prior to subcutaneous administration.

[00367] Once vaccinated, the animals will be released back into their enclosures at Lone Pine Koala Sanctuary.

Sample Collection

[00368] Samples will be collected from the animals at 6 and 12 week time points post- vaccination. The samples collected will include whole blood samples - for plasma and lymphocyte analyses as well as swabs from ocular and penile sites.

Enzyme linked immunosorbent assay (ELISA)

[00369] Enzyme linked immunosorbent assays (ELISAs) will be performed using peptides 2 and 4 on the plasma samples at the 6 and 12 week time points as per Kollipara A. et al. Vaccine 2012, 30:1875-1885, incorporated herein by reference. Measurement of anti-MOMP/anti-Peptide 1 and 3 lymphocyte proliferation response

[00370] Lymphocyte proliferation responses will be assayed as per Khan, S.A., et al. Vaccine 2014 7;32(44):5781-6. Peripheral blood mononuclear cells (PBMCs) will be collected from the whole blood samples collected at the 6 and 12 week time points. PBMCs will be isolated on Ficoll gradients, labelled with carboxyfluorescein succinimidylester (CFSE) then stimulated with peptides 2 and 4 as a positive stimulator, and no antigen as a negative control. Proliferation of PBMCs will be determined using a Beckman Coulter flow cytometer (FC500, Gladesville, NSW, Australia).

RESULTS EXPECTED

[00371] It is expected that the peptide -based vaccine will be safe and that none of the animals will have any adverse reactions to the vaccines, either at the time of administration or in the weeks afterwards.

Expected Antibody Responses at week 6

[00372] It is expected that all animals vaccinated with peptides 2 and 4 will respond by making antibodies (detected in a peptide-specific ELISA) in their plasma at 6 weeks post-vaccination to epitopes 28 and 44. As with Example 2, a stronger antibody response may be seen to one of the epitopes relative to the other epitope.

Expected Antibody Responses at week 12

[00373] It is expected that at week 12, a stronger antibody response to one of the epitopes relative to the other may become evident.

DISCUSSION

[00374] As mentioned in Example 2, previous work has shown that animals vaccinated with a 3-dose ISC adjuvant combined with a full length recombinant MOMP protein produces an antibody and cell mediated immune response. When examined, the specificity of this immune response (antibody response) was directed to specific epitopes along the MOMP protein, and these responses were different to those found in animals which had a natural infection (which they could not control/had not mounted an effective immune response to). The inventor therefore designed a series of short peptides from the full length MOMP protein. The design of these peptides involved the selection of epitopes that reacted in previous MOMP trials, combined with knowledge of amino acid sequences that could contribute both B cell and T cell epitopes. The peptides were further refined as a result of the chemical synthesis process. Of the peptides designed, two were tested in the vaccine trial described in Example 2.

[00375] Even though the peptides were relatively short and even though they were not conjugated to a carrier (to enhance their immunogenicity), by 12 weeks post-vaccination all five Group II animals vaccinated with the two peptides of Example 2 had produced peptide -specific IgG antibodies in their plasma.

[00376] It is expected that a similar conclusion would be reached from the vaccination of animals with peptides 2 and 4.

[00377] The disclosure of each nucleotide and amino acid sequence, patent and scientific document, computer program and algorithm referred to in this specification is incorporated herein by reference in its entirety.

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