WO2015123728A1

WO2015123728A1 - Mycoplasma surface proteins and uses thereof

Info

Publication number: WO2015123728A1
Application number: PCT/AU2015/050061
Authority: WO
Inventors: Veronica M. JAROCKI; Steven P. Djordjevic
Original assignee: University Of Technology, Sydney
Priority date: 2014-02-18
Filing date: 2015-02-18
Publication date: 2015-08-27

Abstract

Immunogenic fragments of a Mycoplasma XAA aminopeptidase are provided as are mutant aminopeptidase proteins having at least partly inactivated active sites and/or metal binding residues. Also provided are antibodies which bind and inhibit Mycoplasma XAA aminopeptidase and methods of detecting, preventing and/or treating Mycoplasma infections in animals and methods of treating Mycoplasma contamination of cell cultures.

Description

TITLE

MYCOPLASMA SURFACE PROTEINS AND USES THEREOF TECHNICAL FIELD

THIS INVENTION relates to Mycoplasma surface proteins. More particularly, this invention relates to isolated immunogenic fragments of surface proteins from Mycoplasma hyopneumoniae and/or Mycoplasma pneumoniae that may be useful in the prevention or treatment of a M. hyopneumoniae- and/or M. pneumonia-associated disease, disorder or condition and/or in the screening, designing and/or engineering of inhibitors of said surface proteins.

BACKGROUND

Mycoplasmas are a genus of bacteria that primarily survive as mucosal pathogens or parasites, typically residing extracellularly in close association with their host's epithelial cells. These bacteria usually occupy the mucosa of the respiratory or urogenital tracts. Given that attachment of the microbe and subsequent initiation of local epithelial damage at the cellular and subcellular levels are primarily responsible for mycoplasma diseases, one logical preventative and/or therapeutic strategy for such diseases is to design a vaccine to prevent microbial attachment and thereby prevent disease initiation.

To this end, surface accessible proteins play a central role in interactions between pathogenic bacteria and their hosts. These molecules engage the host immune system and are fundamental to the ability of the pathogen to survive and proliferate in the host. It is therefore not surprising that many vaccine formulations comprise proteins derived from the cell surface of the pathogen being targeted. Consequently knowledge of surface accessible proteins on microbial pathogens is central to developing efficacious vaccines.

M. hyopneumoniae (Mhp) infection is widespread in farmed pigs throughout the world resulting in major economic losses including reductions in feed efficiency and average daily gain. Mhp is an important component of swine respiratory diseases such as porcine enzootic pneumonia (PEP), porcine respiratory disease complex (PRDC), porcine circovirus type 2-associated post-weaning multi-systemic wasting syndrome, and porcine reproductive and respiratory syndrome virus-induced pneumonia. Hence, the successful control or elimination of Mhp infection is highly desirable. Respiratory diseases of pigs are estimated to cost producers approximately $4-8 per pig.

Current management of Mhp infection relies on the widespread administration of bacterin vaccine formulations that inhibit colonization of the respiratory tract cilia by Mhp along with strategic antibiotic therapy. Commercial vaccines, consisting of inactivated, adjuvant containing whole-cell preparations are manufactured and sold by Boehringer Ingelheim, Zoetis (formerly Pfizer Animal Health) and Merck Animal Health, are widely used worldwide. In many countries, more than 70% of the pig herds are vaccinated against Mhp resulting in improvement of daily weight gain and feed conversion ratio, and reduced mortality rate with an overall reduction in the herd infection level. However, the efficacy of current bacterin vaccines remains in question. Recent studies have shown that, while colonisation of the ciliated respiratory epithelial cells by Mhp is a pre-requisite for the development of mycoplasma pneumonia neither vaccination nor antibiotic therapy can prevent this colonisation. Moreover, studies have demonstrated that vaccination alone with the current bacterin vaccines is not sufficient to eliminate Mhp from infected pig herds.

Mycoplasma pneumoniae {Mpn) is a pathogenic mycoplasma responsible for respiratory tract infections in humans, which occurs worldwide in children and adults. In addition, this microbe may cause a wide array of extra-pulmonary infections and post-infectious complications. Furthermore, Mpn infections have been implicated in the pathogenesis as well as in exacerbation of asthma in children and adults.

Mpn possesses a specialized terminal attachment organelle which enables it to bind to molecules on the surface of the respiratory epithelium. This attachment to the respiratory mucosa places the organism in close association with the host cells and thereby enables secretion of various chemical mediators, causing vacuolation, ciliostasis and exfoliation of mucosal cells. Furthermore, these mediators stimulate host cell production of pro-inflammatory cytokines and lymphocyte activation further damaging the respiratory mucosa and eliciting acute inflammation which results in the characteristic symptoms of Mpn infection.

Antibiotics, in particular macrolides, have historically been the treatment of choice for Mpn infections in humans, but the emergence of clinically significant drug resistance worldwide has precipitated a re-evaluation of their use and a consideration of alternative treatments and preventative therapies such as vaccines. To this end, current inactivated Mpn vaccines may reduce the total rates of both pneumonia and respiratory infections by only -40% (Linchevski et ah, Vaccine, 2009), suggesting that more effective vaccines designed to protect against such infections are required.

SUMMARY

The present invention is predicated in part on the surprising discovery that a Xaa-Pro aminopeptidase (XAP) previously thought to be located intracellulary is present on the surface of Mycoplasma cells.

Accordingly, one form of the invention is broadly directed to immunogenic fragments and/or mutants of a Mycoplasma XAP and their use in preventing and/or treating a Mycoplasma-SLSSOc^' ted disease, disorder or condition.

In a first aspect, the invention provides an immunogenic fragment of an isolated XAP of Mycoplasma.

Preferably, the isolated XAP comprises an amino acid sequence set forth in SEQ ID NO: 2.

In one embodiment, the immunogenic fragment comprises an active aminopeptidase site of the isolated XAP.

In a particular embodiment, the immunogenic fragment comprises, consists or consists essentially of an amino acid sequence set forth in SEQ ID NO: 1.

In another embodiment, the immunogenic fragment comprises one or more metal binding residues of an isolated XAP, such as set forth in SEQ ID NO:2.

In particular embodiments, the one or more metal binding residues include at least one of: an aspartate (D) residue; a glutamate (E) residue; and a histidine (H) residue.

In particular embodiments, the one or more metal binding residues of the isolated XAP or fragment thereof have been substituted or deleted and/or one or more amino acids of the aminopeptidase active site have been substituted or deleted.

In one embodiment, the invention resides in an isolated immunogenic protein comprising one or a plurality of isolated immunogenic fragments of this aspect and one or more heterologous sequences.

This aspect also provides variants and derivatives of the immunogenic fragment or the isolated protein.

In a second aspect, the invention provides an isolated protein comprising an amino acid sequence of an isolated XAP of Mycoplasma or an immunogenic fragment thereof, wherein one or more metal binding residues of the isolated XAP or fragment thereof have been substituted or deleted or wherein one or more amino acids of the the active aminopeptidase site have been substituted or deleted.

In one embodiment, the isolated XAP comprises the amino acid sequence set forth in SEQ ID NO:2.

In one embodiment, the immunogenic fragment of the isolated XAP comprises the amino acid sequence set forth in SEQ ID NO: 1.

In particular embodiments, the one or more metal binding residues include at least one of: an aspartate (D) residue; a glutamate (E) residue; and a histidine (H) residue

This aspect also provides fragments, variants and derivatives of the isolated protein.

In a third aspect, the present invention provides an isolated nucleic acid that comprises a nucleotide sequence that encodes the immunogenic fragment or protein of the first aspect or the isolated protein of the second aspect, or fragments, variants or derivatives thereof, or a nucleotide sequence complementary thereto.

In particular embodiments, the nucleic acid is cDNA.

In a fourth aspect, the invention provides a genetic construct comprising the isolated nucleic acid of the third aspect; operably linked or connected to one or more regulatory sequences in an expression vector.

In a fifth aspect, the invention provides a host cell transformed or transfected with an isolated nucleic acid of the third aspect or a genetic construct of the fourth aspect.

In a sixth aspect, the invention provides a method of producing an isolated immunogenic fragment or protein of the first aspect and/or the isolated protein of the second aspect, comprising; (i) culturing the transformed host cell of the fifth aspect; and (ii) isolating said fragment or protein from said host cell cultured in step (i).

In a seventh aspect, the invention provides an antibody or antibody fragment which binds and/or is raised against an immunogenic fragment and/or isolated protein of the first aspect and/or the second aspect.

Suitably, said antibody or antibody fragment specifically binds said immunogenic fragment and/or said isolated protein.

In an eighth aspect, the invention provides a composition for preventing or treating a Mycoplasma-assoc^' ted disease, disorder or condition, comprising (i) one or more isolated immunogenic fragments and/or proteins of the first aspect; (ii) one or more isolated proteins of the second aspect; (iii) one or more isolated nucleic acids of the third aspect; (iv) one or more genetic constructs of the fourth aspect; (v) one or more host cells of the fifth aspect and/or (v) one or more antibodies or antibody fragments that bind or are raised against an Xaa-Pro aminopeptidase or one or more antibodies or antibody fragments of the seventh aspect; together with a pharmaceutically-acceptable diluent, carrier or excipient.

In an embodiment, the composition is an immunogenic composition.

Preferably, the composition is a vaccine or suitable for immunizing a patient.

In a ninth aspect, the invention provides a method of immunizing an animal including the step of administering (i) one or more isolated immunogenic fragments and/or proteins of the first aspect; (ii) one or more isolated proteins of the second aspect; (iii) one or more isolated nucleic acids of the third aspect; (iv) one or more genetic constructs of the fourth aspect; (v) one or more host cells of the fifth aspect; (v) one or more antibodies or antibody fragments that bind or are raised against an Xaa-Pro aminopeptidase or one or more antibodies or antibody fragments of the seventh aspect; and/or (vi) a composition of the eighth aspect to an animal to thereby induce immunity to Mycoplasma in the animal.

In a tenth aspect, the invention provides a method of treating a Mycoplasma- associated disease, disorder or condition, including the step of administering (i) one or more isolated immunogenic fragments and/or proteins of the first aspect; (ii) one or more isolated proteins of the second aspect; (iii) one or more isolated nucleic acids of the third aspect; (iv) one or more genetic constructs of the fourth aspect; (v) one or more host cells of the fifth aspect; (v) one or more antibodies or antibody fragments that bind or are raised against an Xaa-Pro aminopeptidase or one or more antibodies or antibody fragments of the seventh aspect; and/or (vi) a composition of the eighth aspect to an animal in need thereof to thereby treat the disease, disorder or condition in the animal.

In an embodiment, the composition is an immunogenic composition.

Preferably, the composition acts to enhance or elicit an immune response in said animal to prophylactically or therapeutically treat the Mycoplasma-assocm^' ted disease, disorder or condition.

Preferably, said immune response is a protective immune response and/or confers passive immunity.

In an eleventh aspect, the invention provides a method of detecting Mycoplasma in a biological sample obtainable from an animal, said method including the step of detecting a cell surface-expressed XAP protein on one or more Mycoplasma cells in the biological sample.

In particular embodiments, the XAP protein is detected in the biological sample by binding an antibody or antibody fragment thereto. In an embodiment, the antibody or antibody fragment is that of the seventh aspect. In another embodiment, the antibody or antibody fragment binds or is raised against a substantially full length and/or wild-type XAP.

Suitably, the animal of the eighth, ninth, tenth and eleventh aspects of the invention is a mammal.

Preferably, the mammal is a pig or a human.

In an twelfth aspect, the invention provides a method of detecting, inhibiting and/or preventing Mycoplasma growth and/or activity in vitro, including the step of applying an effective amount of an isolated protein of the second aspect and/or one or more antibodies or antibody fragments that bind or are raised against an Xaa-Pro aminopeptidase or one or more antibodies or antibody fragments of the seventh aspect to an in vitro substrate to thereby detect, inhibit and/or prevent Mycoplasma growth and/or activity in the substrate.

Suitably, the effective amount is a bactericidally and/or a bacteriostatically effective amount.

In a thirteenth aspect, the invention provides a method of identifying, designing and/or engineering of an inhibitor of a Mycoplasma XAP of said method including the steps of:

(i) contacting a Xaa-Pro aminopeptidase protein or a fragment, variant or derivative thereof with a candidate inhibitor; and

(ii) determining whether the candidate inhibitor at least partly reduces, eliminates, suppresses or inhibits an activity of the XAP.

In one embodiment, the activity of the XAP is aminopeptidase activity.

In another embodiment, the activity of the XAP is an ability to bind one or more molecules or atoms, such as a substrate molecule or a metal or metal ion.

Preferably, the candidate inhibitor is an antibody or a small organic molecule.

In a fourteenth aspect, the invention provides an inhibitor of a XAP of a Mycoplasma spp. identified, designed and/or engineered by the method of the thirteenth aspect. Suitably, the inhibitor is for use in the methods of the ninth, tenth, eleventh and twelfth aspects.

In certain embodiments of the aforementioned aspects, "Mycoplasma" includes Mycoplasma hyopneumoniae (Mph) and/or Mycoplasma pneumoniae(Mpri) .

BRIEF DESCRIPTION OF FIGURES

Figure 1. Peptide sequence of XAP (MHJ_0659; UniProt Q4A929). The aminopeptidase site or active site of the XAP protein is underlined. The metal binding amino acid residues of this site are further highlighted and bolded.

Figures 2-6. Tryptic peptides matching to XAP protein from individual surface shaving and LC-MS/MS experiments. Each of Figures 2 to 6 represents a separate biological and technical experimental replicate.

Figure 7. Tryptic peptides matching to XAP (MHJ_0659) from combined surface proteome analyses. Peptide sequences in bold (red) were recovered from shaving freshly cultured Mhp cells with trypsin.

Figure 8. Tryptic peptides matching to XAP protein from surface shaving and avidin chromatography of tryptic peptides from biotinylated XAP and LC-MS/MS experiments. Peptide sequences in bold (red) were recovered from shaving biotin- labelled Mhp cells with trypsin.

Figure 9. Anti-XAP antibodies reduce the efficiency of the enzymatic activity of XAP on Bradykinin. The mass spectrometry experiments performed include: (A) Bradykinin alone; (B) anti-XAP sera alone; (C) XAP + Bradykinin (1 :20 ratio); and (D) XAP + Bradykinin (1 :20 ratio) + anti-XAP sera.

BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO: 1 = peptide sequence of the aminopeptidase or active site of the XAP protein (MHJ_0659; UniProt Q4A929) of Figure 1 as underlined therein (amino acid residues 190-325; total = 136 amino acids); sequence excludes amino acid residues 1- 189 and 326-345.

SEQ ID NO: 2 = peptide sequence of the full length XAP protein (MHJ 0659; UniProt Q4A929) of Figure 1 (total = 345 amino acids).

DETAILED DESCRIPTION

The present invention arises, in part, from the identification of surface accessible XAP proteins of Mhp, the main causative agent of PEP and a major pathogen of swine worldwide inflicting losses of billions of dollars per annum, and Mpn, which is a major pathogen of humans. Relevantly, the candidate proteins are not traditionally thought to be surface exposed on Mhp and Mpn, and thus the suitability of these proteins as immunogens and vaccine candidates is not foreseen. Therefore in one particular form, the invention provides one or more isolated immunogenic proteins, or immunogenic fragments thereof, to prevent or treat a Mhp and/or Mpn related disease, disorder or condition in a mammal, such as a human or pig. In another form, the invention relates to screening, designing or producing an inhibitor of the XAP. Although not wishing to be bound by any particular theory, it is proposed that the aminopeptidase activity of the XAP may be involved in disease pathogenesis. Therefore, elimination or reduction of this activity such as by generation of an immune response to the aminopeptidase active site or the metal-binding regions thereof and/or by the administration of inhibitors of this activity (e.g small organic molecules or monoclonal antibodies) may have efficacy in treating or preventing Mycoplasma-associaXed diseases, disorders or conditions.

Throughout this specification, unless otherwise indicated, "comprise", "comprises" and "comprising" are used inclusively rather than exclusively, so that a stated integer or group of integers may include one or more other non-stated integers or groups of integers.

It will also be appreciated that the indefinite articles "a" and "an" are not to be read as singular indefinite articles or as otherwise excluding more than one or more than a single subject to which the indefinite article refers. For example, "a" protein includes one protein, one or more proteins or a plurality of proteins.

By "consist essentially of is meant in this context that the isolated protein or each immunogenic fragment has one, two or no more than three amino acid residues in addition to the recited amino acid sequence. The additional amino acid residues may occur at the N- and/or C-termini of the recited amino acid sequence, although without limitation thereto.

As generally used herein "Mycoplasma" includes and encompasses organisms of the genus Mycoplasma. Non- limiting examples of Mycoplasma or Mycoplasma spp. that may at least partly cause or initiate a Mycoplasma-associated disease, disorder or condition include M. fermentans, M. orale, M. hominis, M. pulmonis, M. alvi, M. sualvi, M. iowae, M. moatsii, M. pirum, M. buccale, M. spermatophilum, M. pneumoniae, M. salvarium, M. hominis, M. hyopneumoniae, M. penetrans, M. hyorhinis, M. muris, M. fastidiosum, M. amphoriforme, M. gallisepticum, M. genitalium, M. imitans, M. testudinis M. arthritidis and Ureaplasma urealyticum. In certain preferred embodiments, Mycoplasma or Mycoplasma spp. includes Mycoplasma hyopneumoniae (Mph) and/or Mycoplasma pneumoniae (Mpn) including and encompassing all serotypes and strains of Mhp and Mpn respectively, as are known in the art.

For the purposes of this invention, by "isolated' is meant material that has been removed from its natural state or otherwise 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 may be in native, chemical synthetic or recombinant form.

By "protein" is meant an amino acid polymer. The amino acids may be natural or non-natural amino acids, D- or L-amino acids as are well understood in the art.

The term "protein" includes and encompasses "peptide", which is typically used to describe a protein having no more than fifty (50) amino acids and "polypeptide" , which is typically used to describe a protein having more than fifty (50) amino acids.

A "fragment" is a segment, domain, portion or region of a protein, which constitutes less than 100% of the amino acid sequence of the protein.

In general, fragments may comprise up to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250 or 300 amino acids of an amino acid sequence.

In particular embodiments, an immunogenic fragment of an isolated XAP comprises or consists of between 10 and 140 amino acids, more preferably between 15 and 130 amino acids and even more preferably up to 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 or 125 amino acids of the isolated XAP, such as set forth in SEQ ID NO:2.

In a particular embodiment, the immunogenic fragment comprises, consists or consists essentially of an amino acid sequence set forth in SEQ ID NO: 1, which essentially comprises the aminopeptidase active site of the XAP.

In another embodiment, the immunogenic fragment comprises one or more metal binding residues of an isolated XAP, such as set forth in SEQ ID NO: l and SEQ ID O:2.

In particular embodiments, the one or more metal binding residues include at least one of: an aspartate (D) residue; a glutamate (E) residue; and a histidine (H) residue. For the XAP amino acid sequence set forth in SEQ ID NO: 2, the residues are: 190H, 207D, 218D, 282H, and 324E as highlighted in Figure 1.

Thus the immunogenic fragment may comprise some or all of the aminopeptidase active site corresponding to SEQ ID NO: l and constituting a fragment of SEQ ID NO:2, or the immunogenic fragment may comprise a fragment of this active site sequence that comprises at least one of the metal-binding residues thereof.

In the context of the present invention, the term "immunogenic" as used herein indicates the ability or potential of a protein to generate an immune response, such as to Mhp and/or Mpn or molecular components thereof, upon administration of the protein to an animal. It is envisaged that the immune response may be either B- lymphocyte or T-lymphocyte mediated, or a combination thereof. Advantageously, by "immunogenic" is meant capable of eliciting a B-lymphocyte response, although is not limited thereto. "Immunogenic" can also mean capable of eliciting a neutralising antibody response.

The invention also provides an isolated protein comprising one or more immunogenic fragments of the XAP. Suitably, the isolated protein is not full length or wild-type XAP.

In one particular embodiment, the invention contemplates an isolated protein comprising a plurality of immunogenic fragments described herein, such as in the form of a "poly tope" protein. For example, said immunogenic fragments may be present singly or as repeats, which also includes tandemly repeated fragments. Heterologous amino acid sequences (e.g "spacer" amino acids) may also be included between one or a plurality of the immunogenic fragments present in said isolated protein.

In a further aspect, the invention resides in an isolated protein comprising an amino acid sequence of an XAP or fragment, variant or derivative thereof, and comprising at least one amino acid substitution or deletion of at least one of the metal binding residues of an XAP of Mycoplasma.

It is proposed that the metal binding residues of an XAP may play a pivotal role in the enzyme's activity, as XAP, like all metalloaminopeptidases, liberates amino acids from the N-terminus of peptides and proteins via a cleavage event mediated by a water molecule that is activated by a divalent metal cation. The Mhp metalloaminopeptidases belong to protease families that utilise two co-catalytic divalent cations. These two metal ions are pentrahedrally coordinated by three amino acid side chains and a water molecule. The known ligands for metalloaminopeptidases are H, E and D acids, lysine (K) and arginine (R). For the XAP sequence set forth in SEQ ID NO:2, the predicted amino acids that bind the two metal ions needed to exert aminopeptidase activity are: 190H, 207D, 218D, 282H, and 324E as highlighted in Figure 1. These residues are generally conserved among XAPs.

Accordingly, the isolated protein of this aspect, or a variant, fragment or derivative thereof, may act as a dominant negative mutant of an XAP. As used herein, the term "dominant negative mutant" refers to a mutant polypeptide or protein, which lacks wild-type activity and when expressed or present intracellularly or extracellularly wherein a wild-type of the same polypeptide or protein is also expressed or present, dominates the wild-type polypeptide or protein and effectively competes with the wild-type version for substrates, receptors, ligands, etc., and thereby inhibits or blocks the activity of the wild type polypeptide or protein.

The invention also provides variants of the isolated immunogenic fragments and/or proteins described herein.

As used herein, a protein "variant" shares a definable nucleotide or amino acid sequence relationship with an isolated protein or immunogenic fragment disclosed herein. Preferably, protein variants share at least 35% or 40%, preferably at least 45% or 50% or more preferably at least 55%, 60% or 65% or even more preferably 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with an amino acid sequence of the invention, such as the amino acid sequence set forth in SEQ ID NO: 1.

The variant" proteins or fragments disclosed herein have one or more amino acids deleted or substituted by different amino acids. It is well understood in the art that some amino acids may be substituted or deleted without changing the activity of the immunogenic fragment and/or protein (conservative substitutions).

The term "variant" also includes isolated proteins or fragments thereof disclosed herein, produced from, or comprising amino acid sequences of, naturally occurring (e.g., allelic) variants, orthologs {e.g., from a species other than Mycoplasma hyopneumoniae) and synthetic variants, such as produced in vitro using mutagenesis techniques.

Variants may retain the biological activity of a corresponding wild type protein {e.g. allelic variants, paralogs and orthologs) or may lack, or have a substantially reduced, biological activity compared to a corresponding wild type protein. Terms used generally herein to describe sequence relationships between respective proteins and nucleic acids include "comparison window", "sequence identity", "percentage of sequence identity" and "substantial identity" . Because respective nucleic acids/proteins may each comprise (1) only one or more portions of a complete nucleic acid/protein sequence that are shared by the nucleic acids/proteins, and (2) one or more portions which are divergent between the nucleic acids/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 6, 9 or 12 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 for optimal alignment of the respective sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (Geneworks program by Intelligenetics; GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA, incorporated herein by reference) or by inspection and the best alignment {i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res. 25 3389, which is incorporated herein by reference. A detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (lohn Wiley & Sons Inc NY, 1995-1999).

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, I) 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'¹ 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).

Derivatives of the immunogenic fragments and/or proteins are also provided.

As used herein, "derivative" proteins have been altered, for example by conjugation or complexing with other chemical moieties, by post-translational modification (e.g. phosphorylation, acetylation and the like), modification of glycosylation (e.g. adding, removing or altering glycosylation) and/or inclusion of additional amino acid sequences as would be understood in the art.

Additional amino acid sequences may include fusion partner amino acid sequences which create a fusion protein. By way of example, fusion partner amino acid sequences may assist in detection and/or purification of the isolated fusion protein. Non-limiting examples include metal-binding (e.g. polyhistidine) fusion partners, maltose binding protein (MBP), Protein A, glutathione S-transferase (GST), fluorescent protein sequences (e.g. GFP), epitope tags such as myc, FLAG and haemagglutinin tags.

Other derivatives contemplated by the invention include, but are not limited to, modification to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide, or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the immunogenic proteins, fragments and variants of the invention.

In this regard, the skilled person is referred to Chapter 15 of CURRENT PROTOCOLS IN PROTEIN SCIENCE, Eds. Coligan et al. (John Wiley & Sons NY 1995-2008) for more extensive methodology relating to chemical modification of proteins.

The isolated immunogenic proteins, fragments and/or derivatives of the present invention may be produced by any means known in the art, including but not limited to, chemical synthesis, recombinant DNA technology and proteolytic cleavage to produce peptide fragments. Chemical synthesis is inclusive of solid phase and solution phase synthesis. Such methods are well known in the art, although reference is made to examples of chemical synthesis techniques as provided in Chapter 9 of SYNTHETIC VACCINES Ed. Nicholson (Blackwell Scientific Publications) and Chapter 15 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al, (John Wiley & Sons, Inc. NY USA 1995-2008). In this regard, reference is also made to International Publication WO 99/02550 and International Publication WO 97/45444.

Recombinant proteins and immunogenic fragments 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), in particular Sections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al, (John Wiley & Sons, Inc. NY USA 1995-2008), in particular Chapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al, (John Wiley & Sons, Inc. NY USA 1995-2008), in particular Chapters 1, 5 and 6.

Alternatively, fragments can be produced by digestion of a polypeptide, such as a XAP, with proteinases such as endoLys-C, endoArg-C, endoGlu-C and V8- protease. The digested fragments can be purified by chromatographic techniques as are well known in the art.

In another aspect, the present invention contemplates isolated nucleic acids that encode, or are complementary to nucleic acid sequence which encodes, the immunogenic fragments and isolated proteins disclosed herein.

Nucleotide sequences encoding the isolated immunogenic proteins, isolated immunogenic fragments, variants, derivatives and polytopes of the invention may be readily deduced from the complete genomic nucleic acid sequence of either Mhp, published for example in Minion et al , J Bacteriol, Nov 2004; 186(21):7123-7133 (GenBank Accession No. AE017332), or Mpn, published for example in Dandekar et al, Nucl Acids Res, 2000; 28(17):3278-3288 (GenBank Accession No. U00089), although without limitation thereto.

This aspect also includes fragments, variants and derivatives of said isolated nucleic acid.

The term "nucleic acicT as used herein designates single- or double-stranded DNA and RNA. DNA includes genomic DNA and cDNA. RNA includes mRNA, RNA, RNAi, siRNA, cRNA and autocatalytic RNA. Nucleic acids may also be DNA- RNA hybrids. A nucleic acid comprises a nucleotide sequence which typically includes nucleotides that comprise an A, G, C, T or U base. However, nucleotide sequences may include other bases such as inosine, methylycytosine, methylinosine, methyladenosine and/or thiouridine, although without limitation thereto.

Accordingly, in particular embodiments, the isolated nucleic acid is cDNA.

A "polynucleotide " is a nucleic acid having eighty (80) or more contiguous nucleotides, while an "oligonucleotide " has less than eighty (80) contiguous nucleotides.

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

A "primer" is usually a single-stranded oligonucleotide, preferably having 15- 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™.

Another particular aspect of the invention provides a variant of an isolated nucleic acid that encodes an isolated immunogenic fragment or protein of the invention.

In one embodiment, nucleic acid variants encode a variant of an isolated protein of the invention.

In another embodiment, nucleic acid variants share at least 35%, 40%, 45%, 50%, 55%, 60% or 65%, 66%, 67%, 68%, 69%, preferably at least 70%, 71%, 72%, 73%, 74% or 75%, more preferably at least 80%, 81%, 82%, 83%, 84%, or 85%, and even more preferably at least 90%, 91%, 92%, 93%, 94%, or 95% nucleotide sequence identity with an isolated nucleic acid of the invention.

The present invention also contemplates nucleic acids that have been modified such as by taking advantage of codon sequence redundancy. In a more particular example, codon usage may be modified to optimize expression of a nucleic acid in a particular organism or cell type.

The invention further provides use of modified purines (for example, inosine, methylinosine and methyladenosine) and modified pyrimidines (for example, thiouridine and methylcytosine) in nucleic acids of the invention.

It will be well appreciated by a person of skill in the art that the isolated nucleic acids of the invention can be conveniently prepared using standard protocols such as those described in Chapter 2 and Chapter 3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Eds. Ausubel et al. John Wiley & Sons NY, 1995-2008).

In yet another embodiment, complementary nucleic acids hybridise to nucleic acids of the invention under high stringency conditions.

"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.

"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.

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

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.

Complementary nucleotide sequences may be 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, typically using a labelled probe or other complementary nucleic acid. 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. 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. 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 hybridization. Other typical examples of this procedure are described in Chapters 8-12 of Sambrook et al, MOLECULAR CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989).

Methods for detecting labelled nucleic acids hybridized to an immobilized nucleic acid are well known to practitioners in the art. Such methods include autoradiography, chemiluminescent, fluorescent and colorimetric detection.

Nucleic acids may also be isolated, detected and/or subjected to recombinant DNA technology using nucleic acid sequence amplification techniques.

Suitable nucleic acid amplification techniques covering both thermal and isothermal methods are well known to the skilled addressee, and include polymerase chain reaction (PCR); strand displacement amplification (SDA); rolling circle replication (RCR); nucleic acid sequence-based amplification (NASBA), Q-β replicase amplification, recombinase polymerase amplification (RPA) and helicase- dependent amplification, although without limitation thereto.

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

Nucleic acid amplification techniques may include particular quantitative and semi-quantitative techniques such as qPCR, real-time PCR and competitive PCR, as are well known in the art.

In another aspect, the invention provides a genetic construct comprising: (i) the isolated nucleic acid described herein; or (ii) an isolated nucleic acid comprising a nucleotide sequence complementary thereto; operably linked or connected to one or more regulatory sequences in an expression vector.

Suitably, the genetic construct is in the form of, or comprises genetic components of, a plasmid, bacteriophage, a cosmid, a yeast or bacterial artificial chromosome as are well understood in the art. Genetic constructs may be suitable for maintenance and propagation of the isolated nucleic acid in bacteria or other host cells, for manipulation by recombinant DNA technology and/or expression of the nucleic acid or an encoded protein of the invention.

For the purposes of host cell expression, the genetic construct is an expression construct. Suitably, the expression construct comprises the nucleic acid of the invention operably linked to one or more additional sequences in an expression vector. An "expression vector¹' may be either a self-replicating extra-chromosomal vector such as a plasmid, or a vector that integrates into a host genome. By "operably linked' is meant that said additional nucleotide sequence(s) is/are positioned relative to the nucleic acid of the invention preferably to initiate, regulate or otherwise control transcription.

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.

Typically, said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences.

Constitutive or inducible promoters as known in the art are contemplated by the invention.

The expression construct may also include an additional nucleotide sequence encoding a fusion partner (typically provided by the expression vector) so that the recombinant allergenic protein of the invention is expressed as a fusion protein, as hereinbefore described.

In a further aspect, the invention provides a host cell transformed with a nucleic acid molecule or a genetic construct described herein.

Suitable host cells for expression may be prokaryotic or eukaryotic. For example, suitable host cells may include but are not limited to mammalian cells {e.g. HeLa, HEK293T, Jurkat cells), yeast cells (e.g. Saccharomyces cerevisiae), insect cells (e.g. Sf9, Trichoplusia ni) utilized with or without a baculovirus expression system, plant cells (e.g. Chlamydomonas reinhardtii, Phaeodactylum tricornutum) or bacterial cells, such as E. coli. Introduction of genetic constructs into host cells (whether prokaryotic or eukaryotic) is well known in the art, as for example described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al, (John Wiley & Sons, Inc. 1995-2009), in particular Chapters 9 and 16.

In yet another aspect, the invention provides a method of producing an isolated immunogenic fragment or isolated protein described herein, comprising; (i) culturing the previously transformed host cell hereinbefore described; and (ii) isolating said fragment or protein from said host cell cultured in step (i).

The recombinant protein may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook, et al., MOLECULAR CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989), in particular Sections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al, (John Wiley & Sons, Inc. 1995-2009), in particular Chapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al, (John Wiley & Sons, Inc. 1995-2009), in particular Chapters 1, 5 and 6.

In a further aspect, the invention provides an antibody or antibody fragment which binds and/or is raised against an immunogenic fragment and/or isolated protein described herein.

Suitably, said antibody or antibody fragment specifically binds said isolated immunogenic fragment and/or protein.

In some embodiments, the antibody may reduce, eliminate, inhibit or suppress the aminopeptidase activity of an XAP of Mycoplasma and/or may inhibit reduce, eliminate, inhibit or suppress binding of XAP to metal ions and/or substrate molecules.

In other embodiments, the antibody or antibody fragment may be used in in vitro and/or cell culture applications, such as for the detection, prevention, elimination or minimization of mycoplasma contamination of cell cultures and the like as described hereinafter. This may involve, but is not limited to, coating filters, plates and other cell culture equipment with an antibody and/or antibody fragment to XAP. Additionally, the antibody or fragment thereof may be included in a treatment solution to be added directly to cells in cell culture with optionally one or more antibiotics, antimetabolic agents etc. that target Mycoplasma. As would be appreciated by the skilled artisan one or more isolated proteins described herein, such as those dominant negative mutant proteins or polypeptides may be utilized in a similar manner.

Antibodies of the invention may be polyclonal or monoclonal, native or recombinant. 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.

Generally, antibodies of the invention bind to or conjugate with an isolated protein, fragment, variant, or derivative of the invention. For example, the antibodies may be polyclonal antibodies. Such antibodies may be prepared for example by injecting an isolated protein, fragment, variant or derivative 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.

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 isolated proteins, fragments, variants or derivatives of the invention.

The invention also includes within its scope antibody fragments, such as Fc, Fab or F(ab)2 fragments of the polyclonal or monoclonal antibodies referred to above. Alternatively, the antibodies may comprise single chain Fv antibodies (scFvs) against the peptides 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 the article by Winter & Milstein, 1991, Nature 349:293, which are incorporated herein by reference. The invention is also contemplated to include multivalent recombinant antibody fragments, so-called diabodies, triabodies and/or tetrabodies, comprising a plurality of scFvs, as well as dimerisation-activated demibodies (e.g., WO/2007/062466). By way of example, such antibodies may be prepared in accordance with the methods described in Holliger et al., 1993 Proc Natl Acad Sci USA 90:6444-6448; or in Kipriyanov, 2009 Methods Mol Biol 562: 177-93 and herein incorporated by reference in their entirety.

Antibodies and antibody fragments of the invention may be particularly suitable for affinity chromatography purification of the isolated immunogenic fragments and/or proteins described herein. For example reference may be made to affinity chromatographic procedures described in Chapter 9.5 of Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY, supra.

In another embodiment, antibodies or antibody fragments that bind or are raised against a substantially full length or wild-type Xaa-Pro aminopeptidase may be used to detect cell surface-expressed Xaa-Pro aminopeptidase and/or for passive immunization against Mycoplasma-associated diseases, disorders or conditions. In particular aspects, the invention provides compositions and/or methods of preventing, treating and/or immunizing against a Mycoplasma-associated disease, disorder or condition in an animal.

As used herein, "treating"^'' (or "treat" or "treatment") refers to a therapeutic intervention that ameliorates a sign or symptom of a Mycoplasma (e.g a Mhp and/or pn)-associated disease, disorder or condition after it has begun to develop. The term "ameliorating " with reference to a Mhp- and/or Mpn -associated disease, disorder or condition, refers to any observable beneficial effect of the treatment. Treatment need not be absolute to be beneficial to the subject. The beneficial effect can be determined using any methods or standards known to the ordinarily skilled artisan.

As used herein, "preventing" (or "prevent" or "prevention") refers to a course of action (such as administering a composition comprising a therapeutically effective amount of one or more immunogenic proteins and/or a fragment, variant or derivative thereof of the present invention) initiated prior to the onset of a symptom, aspect, or characteristic of a Mhp and/or Mpn -associated disease, disorder or condition, so as to prevent or reduce the symptom, aspect, or characteristic. It is to be understood that such preventing need not be absolute to be beneficial to a subject. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a Mhp - and/or Mpn -associated disease, disorder or condition, or exhibits only early signs for the purpose of decreasing the risk of developing a symptom, aspect, or characteristic of a Mhp - and/or Mpn -associated disease, disorder or condition.

In the context of the present invention, by "Mhp and/or Mpn -associated disease, disorder or condition " is meant any clinical pathology resulting from infection by Mhp or Mpn.

Typically, Mhp and Mpn colonise the mucosa of the respiratory tract, particularly, although not exclusively, in mammals such as humans and pigs.

Mhp is known to cause PEP, a highly infectious and chronic disease affecting pigs. Diseases and/or clinical symptoms associated with Mhp include pneumonia, pleuritis, pericarditis, reduced growth rate and feed efficiency, dyspnoea, fever, anorexia, septicaemia and Porcine Respiratory Disease Complex (PRDC), although without limitation thereto.

Mpn is a common cause of pneumonia, so-called Mycoplasma pneumonia, and/or bronchitis in humans. Diseases and/or clinical symptoms associated with Mpn infection include pharyngitis, bronchitis, tonsillitis, pneumonia, septicemia, haemolytic anaemia, rheumatoid arthritis, Stevens-Johnson syndrome, encephalitis, Guillain-Barre syndrome and fever, although without limitation thereto.

A composition for preventing or treating a Mycoplasma-a.ssocia.ted disease, disorder or condition may comprise (i) one or more immunogenic fragments and/or proteins described herein; (ii) one or more isolated proteins described herein; (iii) one or more isolated nucleic acids described herein; (iv) one or more genetic constructs described herein; and/or (v) one or more antibodies or antibody fragments that bind or are raised against an Xaa-Pro aminopeptidase (e.g., substantially full length and/or wild-type XAP), an immunogenic fragment or isolated protein such as those described herein, together with a pharmaceutically-acceptable diluent, carrier or excipient.

By "pharmaceutically-acceptable carrier, diluent or excipient" 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 sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates and pyrogen-free water.

A useful reference describing pharmaceutically acceptable carriers, diluents and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991) which is incorporated herein by reference.

In a preferred embodiment, the pharmaceutical composition of the present invention is an immunogenic composition.

More preferably, the immunogenic composition is an immunogenic composition.

In a particular preferred embodiment, the immunogenic composition is a vaccine.

In another particular preferred embodiment, the immunogenic composition comprises one or more antibodies disclosed herein for passive immunization of an animal, inclusive of humans and pigs.

Suitable vaccines may be in the form of proteinaceous vaccines, and in particular, comprise one or more immunogenic fragments of an XAP of Mhp and/or Mpn, or a fragment, variant or derivative thereof as described herein. It will be appreciated by the foregoing that the immunogenic composition and/or vaccine of the invention may include an "immunologically-acceptable carrier, diluent or excipienf.

Useful carriers are well known in the art and include for example: thyroglobulin; albumins such as human serum albumin; toxins, toxoids or any mutant crossreactive material (CRM) of the toxin from tetanus, diphtheria, pertussis, Pseudomonas, E. coli, Staphylococcus, and Streptococcus; polyamino acids such as poly(lysine:glutamic acid); influenza; Rotavirus VP6, Parvovirus 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 T cell epitope of a bacterial toxin, toxoid or CRM may be used. In this regard, reference may be made to U. S. Patent No 5,785,973 which is incorporated herein by reference.

The "immunologically-acceptable carrier, diluent or excipienf includes within its scope water, bicarbonate buffer, phosphate buffered saline or saline and/or an adjuvant as is well known in the art. As will be understood in the art, an "adjuvant" means a composition comprised of one or more substances that enhances the immunogenicity and efficacy of a vaccine composition. Non-limiting examples of suitable adjuvants include squalane and squalene (or other oils of plant or animal origin); block copolymers; detergents such as Tween®-80; Quil® A, mineral oils such as Drakeol or Marcol, vegetable oils such as peanut oil; Corynebacterium-derwed adjuvants such as Corynebacterium parvum Propionibacterium-derived adjuvants such as Propionibacterium acne; Mycobacterium bovis (Bacille Calmette and Guerin or BCG); Bordetella pertussis antigens; tetanus toxoid; diphtheria toxoid; surface active substances such as hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyldioctadecylammonium bromide, NN-dicoctadecyl-N', N'bis(2-hydroxyethyl- propanediamine), methoxyhexadecylglycerol, and pluronic polyols; polyamines such as pyran, dextransulfate, poly IC carbopol; peptides such as muramyl dipeptide and derivatives, dimethylglycine, tuftsin; oil emulsions; and mineral gels such as aluminum phosphate, aluminum hydroxide or alum; interleukins such as interleukin 2 and interleukin 12; monokines such as interleukin 1 ; tumour necrosis factor; interferons such as gamma interferon; combinations such as saponin-aluminium hydroxide or Quil-A aluminium hydroxide; liposomes; ISCOM® and ISCOMATRJX® adjuvant; mycobacterial cell wall extract; synthetic glycopeptides such as muramyl dipeptides or other derivatives; Avridine; Lipid A derivatives; dextran sulfate; DEAE-Dextran alone or with aluminium phosphate; carboxypolymethylene such as Carbopol¹ EMA; acrylic copolymer emulsions such as Neocryl A640 (e.g. U. S. Pat. No. 5,047,238); water in oil emulsifiers such as Montanide ISA 720; poliovirus, vaccinia or animal poxvirus proteins; or mixtures thereof.

With regard to subunit vaccines, an example of such a vaccine may be formulated with ISCOMs, such as described in International Publication W097/45444.

An example of a vaccine in the form of a water-in-oil formulation includes Montanide ISA 720, such as described in International Publication W097/45444.

Any suitable procedure is contemplated for producing vaccine compositions. 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.

Alternatively, a vaccine may be in the form of a nucleic acid vaccine and in particular, a DNA vaccine. A useful reference describing DNA vaccinology is DNA Vaccines, Methods and Protocols, Second Edition (Volume 127 of Methods in Molecular Medicine series, Humana Press, 2006) and is incorporated herein by reference.

One particular broad application of the present invention is provision of methods of treating or immunising an animal by administering a composition, such as that of the present invention, to said animal.

Accordingly, an aspect of the invention provides a method of immunizing an animal including the step of administering a composition, such as that of the present invention, to an animal to thereby induce immunity to a Mycoplasma spp. in said animal. Suitably, immunity to the Mycoplasma spp. prevents the animal contracting a Mycoplasma-associated disease, disorder or condition. Typically, the Mycoplasma spp. is Mhp and/or Mpn. It would be appreciated by the skilled artisan, however, that owing to homology observed in the protein sequence for the aminopeptidase XAP across different Mycoplasma species, the method may also be used to immunise an animal against a further Mycoplasma species, including, but not limited to, those hereinbefore listed.

In another aspect, the invention also provides a method of treating a Mycoplasma-associated disease, disorder or condition, including the step of administering a composition, such as that of the present invention, to an animal in need thereof.

It would be appreciated that compositions for administration in the methods of the two aforementioned aspects may comprise, but are not necessarily limited to, one or more antibodies or one or more antibody fragments of the present invention which have been raised against an immunogenic fragment and/or isolated protein described herein. Accordingly, in certain embodiments, such compositions may comprise one or more antibodies or one or more antibody fragments that may bind or are raised against a substantially full length and/or wild-type XAP protein, such as that set forth in SEQ ID N0 2.

Preferably, the method elicits or enhances an immune response in said animal to prophylactically or therapeutically treat a Mycoplasma-a.ssocia.ted disease, disorder or condition in the animal.

Typically, the Mycoplasma species is M. hyopneumoniae and/or M. pneumonia. Similar to the previous aspect, the method may also be used to treat an animal for a further Mycoplasma species, including, but not limited to, those hereinbefore listed.

Such compositions may be delivered for the purposes of generating at least partial immunity, and preferably protective immunity, or for generating an immune response, preferably a protective immune response, to a Mycoplasma spp., such as Mhp and/or Mpn, upon administration to a host, although without limitation thereto.

By "protective immunity " is meant a level of immunity whereby the responsiveness to an antigen or antigens is sufficient to lead to rapid binding and/or elimination of said antigens and thus prevent a Mycoplasma infection in an animal.

By "protective immune response " is meant a level of immune response that is sufficient to prevent or reduce the severity, symptom, aspect, or characteristic of a current Mycoplasma infection in an animal.

Furthermore, it will also be appreciated that compositions comprising antibodies that bind or are raised against an Xaa-Pro aminopeptidase, immunogenic fragment or isolated protein such as those of the present invention, may be useful for passive immunisation, or for generating a passive immune response, against a Mycoplasma infection. Moreover, such compositions may also be effective in treating a Mycoplasma-associ&ted disease, disorder or condition.

Suitably, the methods of detecting, treating and/or immunizing against Mycoplasma in an animal of the present invention are performed on a mammal.

In one embodiment, the mammal is a pig.

In another embodiment, the mammal is a human.

In alternative embodiments, the isolated immunogenic proteins and/or fragments of the present invention may be used as a vaccine in the purified form, fused to immunogenic carrier proteins, or expressed by live vaccine delivery systems including attenuated viruses, virus-like particles or live attenuated bacteria.

Compositions and vaccines of the invention may be administered to humans in the form of attenuated or inactivated bacteria that may be induced to express one or more isolated immunogenic proteins or immunogenic fragments of the present invention. Non-limiting examples of attenuated bacteria include Salmonella species, for example Salmonella enterica var. Typhimurium or Salmonella typhi. Alternatively, other enteric pathogens such as Shigella species or E. coli may be used in attenuated form. Attenuated Salmonella strains have been constructed by inactivating genes in the aromatic amino acid biosynthetic pathway (Alderton et at, Avian Diseases 35 435), by introducing mutations into two genes in the aromatic amino acid biosynthetic pathway (such as described in U. S. patent 5,770,214) or in other genes such as htrA (such as described in U.S. patent 5,980,907) or in genes encoding outer membrane proteins, such as ompR (such as described in U.S. patent 5,851,519).

Expression of the proteins, peptides, fragments or fusion proteins containing transport or immunogenic functions and could result in production of the immunogenic protein, peptide or fragment in the cytoplasm, cell wall, exposed on the cell surface or produced in a secreted form.

In light of the foregoing, therapeutic application of mRNA-based gene silencing technologies is also contemplated. Useful references describing such technology include RNAi: Design and Application (Methods in Molecular Biology, vol. 442, Humana Press NY. USA, 2008) and RNAi: A Guide to Gene Silencing (Cold Spring Harbor Laboratory Press N.Y. USA, 2003).

By "administering" or "administration" is meant the introduction of a composition disclosed herein into a subject by a particular, chosen route. Any safe route of administration may be employed for providing a patient with the composition of the invention. For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular and transdermal administration may be employed.

Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, nasal sprays, 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 effected 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 effected by using other polymer matrices, liposomes and/or microspheres.

Compositions of the present invention suitable for oral or parenteral administration may be presented as discrete units such as capsules, sachets, functional foods/feeds or tablets each containing a pre-determined amount of one or more therapeutic agents 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 pharmacy but all methods include the step of bringing into association one or more 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.

The above compositions may be administered in a manner compatible with the dosage formulation, and in such amount as is pharmaceutically-effective. The dose administered to a patient, in the context of the present invention, should be sufficient to effect a beneficial response in a patient over an appropriate period of time. The quantity of agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of the practitioner. In another aspect, the invention provides a method of detecting Mycoplasma in a biological sample obtainable from an animal, said method including the step of detecting a XAP protein on an extracellular surface of one or more Mycoplasma cells in the biological sample.

In certain embodiments, the biological sample may be a pathology sample that comprises one or more fluids, cells, tissues, organs or organ samples obtained from an animal. Non-limiting examples include blood, plasma, serum, lymphocytes, urine, faeces, amniotic fluid, cervical samples, cerebrospinal fluid, tissue biopsies, bone marrow, bronchoalveolar lavage fluid, sputum and skin.

In particular embodiments, the XAP protein is detected in the biological sample by binding an antibody or antibody fragment thereto. Preferably, the antibody or antibody fragment binds or has been raised against an Xaa-Pro aminopeptidase . As hereinbefore discussed, the antibody or antibody fragment need not be that of the present invention. In particular preferred embodiments, however, the antibody or antibody fragment is that of the invention hereinbefore described.

In certain embodiments, the XAP protein in the subject is detected in the biological sample by binding a small molecule thereto.

Suitably, detecting XAP includes the step of forming a detectable complex between an antibody, antibody fragment or small molecule and XAP. The complex so formed may be detected by any technique, assay or means known in the art, including immunoblotting, immunohistochemistry, immunocytochemistry, immunofluroescence, immunoprecipitation, ELISA, flow cytometry, magnetic bead separation, and biosensor-based detection systems such as surface plasmon resonance, although without limitation thereto.

To facilitate detection the antibody may be directly labelled or a labelled secondary antibody may be used. Additionally, the small molecule may be directly labelled.

The label may be selected from a group including a chromogen, a catalyst, biotin, digoxigenin, an enzyme, a fluorophore, a chemiluminescent molecule, a radioisotope, a drug, a magnetic bead and/or a direct visual label.

In the case of a direct visual label, use may be made of a colloidal metallic or non-metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like. The fluorophore may be, for example, fluorescein isothiocyanate (FITC), Alexa dyes, tetramethylrhodamine isothiocyanate (TRITL), allophycocyanin (APC), Texas Red, Cy5, Cy3, or R-Phycoerythrin (RPE) as are well known in the art.

The enzyme may be horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase or glucose oxidase, although without limitation thereto.

In some embodiments, detection methods may be performed in "high throughput" diagnostic tests or procedures such as performed by commercial pathology laboratories or in hospitals.

It would be further appreciated, that such detection methods of XAP may have potential utility in characterising disease progression and/or severity of a Mycoplasma-associated disease, disorder or condition in an animal. Additionally, such methods may be used for selecting animals for anti-XAP treatment, such as by a so-called "companion diagnostic".

In a further aspect, the invention provides a method of detecting, inhibiting and/or preventing Mycoplasma growth and/or activity in in vitro, including the step of applying an effective amount of an isolated protein of the second aspect and/or an antibody or antibody fragment of the seventh aspect to a substrate in vitro to thereby detect, inhibit and/or prevent Mycoplasma growth and/or activity in the substrate .

It is well established that Mycoplasma, such as Mycoplasma hyorhinis, Mycoplasma fermentans, Mycoplasma orale, Mycoplasma argininii, Mycoplasma hominis, or Acholeplasma laidlawi, may contaminate cells during cell culture. Such contamination during cell culturing may occur in pharmaceutical companies, hospitals, and academic laboratories where cell culturing is frequently conducted. By way of example, Mycoplasma contamination may occur during cell line construction from a living organism infected with Mycoplasma. Further, user error and carelessness in cell culture technique in laboratories may cause contamination between cell lines, thereby potentially resulting in widespread Mycoplasma contamination.

Mycoplasma contamination in cell culture is typically not accompanied with any visible changes, such as an increase in a turbidity of a medium as in the case of other infection sources (i.e., bacteria having cell wall or fungi), or cell death as in the case of virus. Notwithstanding the lack of visible changes with Mycoplasma contamination in cell culture, such contamination may cause various unpredictable and unwanted outcomes in infected cell lines, such as abnormal gene and protein expression and altered metabolism. Thus, experimenters may fail to recognize Mycoplasma contamination and unknowingly produce abnormal experimental results therefrom. Additionally, Mycoplasma is not affected by penicillin and other beta lactam antibiotics which are typically used in cell culture, owing to their lack of a cell wall.

Accordingly, it would be understood that an in vitro substrate may include any nutrient medium in which cells of any type may be cultured in vitro and any culture supplements or additives, such as serum, glutamine, growth factors and antibiotics, that may be added thereto.

Furthermore, an in vitro substrate may refer to as well as any physical cell culture medium, device or piece of equipment, and in particular single use devices or so called "disposables", for use in cell culture. This may include, for example, flasks, plates, roller bottles, multiwell plates, chamber slides, coverslips, filters, pipettes, cell scrapers, cell lifters, bags for culture media storage, bottles for culture media storage, tips, cryovials, centrifuge tubes, syringes and needles.

It would be appreciated that the effective amount of the antibody, antibody fragment and/or protein may be applied to an in vitro medium, such as a nutrient medium and/or a cell culture device, prior to the medium' s use in an attempt to prevent or limit a Mycoplasma infection in vitro. Alternatively, the antibody, antibody fragment and/or protein may be applied to an in vitro medium, in or on which a Mycoplasma infection has been detected previously.

In embodiments relating to inhibiting and/or preventing the growth and/or activity of the Mycoplasma spp., the effective amount is suitably a bactericidally and/or a bacteriostatically effective amount.

In embodiments relating to detecting a Mycoplasma spp. in vitro, detection may be facilitated by directly labelling the antibody, antibody fragment or protein as hereinbefore described or a labelled secondary antibody may be used. The labelled secondary antibody may be as hereinbefore described.

Suitably, detecting a Mycoplasma spp. in vitro includes the step of forming a detectable complex between the antibody, antibody fragment or protein and XAP. The complex so formed may be detected by any technique, assay or means known in the art, such as those hereinbefore described.

In yet another aspect, the invention provides a method of identifying, designing and/or engineering of an inhibitor of XAP of Mycoplasma, said method including the steps of:

(i) contacting a XAP protein or a fragment, variant or derivative thereof with a candidate inhibitor; and

(ii) determining whether the candidate inhibitor reduces, eliminates, suppresses or inhibits an activity of XAP.

In some embodiments, the inhibitor may at least partly reduce, eliminate, inhibit or suppress the aminopeptidase activity of XAP.

In other embodiments, the inhibitor would act to at least partly reduce, eliminate, inhibit or suppress the ability of XAP to bind to one or more other molecules or atoms, such as a substrate molecule or a divalent metal cation.

Suitably, the inhibitor would possess or display minimal or no significant off- target and/or nonspecific effects.

Preferably, the candidate inhibitor is an antibody or a small organic molecule.

In embodiments relating to antibody inhibitors, the antibody may be polyclonal or monoclonal, native or recombinant, as hereinbefore described. Typically, the inhibitory activity of candidate inhibitor antibodies may be assessed by in vitro and/or in vivo assays that detect or measure aminopeptidase activity of an XAP in the presence of the antibody.

In embodiments relating to small organic molecule inhibitors, this may involve screening of large compound libraries, numbering hundreds of thousands to millions of candidate inhibitors (chemical compounds including synthetic, small organic molecules or natural products, for example) which may be screened or tested for biological activity at any one of hundreds of molecular targets in order to find potential new drugs, or lead compounds. Screening methods may include, but are not limited to, computer-based ("in silico") screening and high throughput screening based on in vitro assays.

Typically, the active compounds, or "hits", from this initial screening process are then tested sequentially through a series of other in vitro and/or in vivo tests to further characterize the active compounds. A progressively smaller number of the "successful" compounds at each stage are selected for subsequent testing, eventually leading to one or more drug candidates being selected to proceed to being tested in human clinical trials.

Drug design and engineering denotes the development of new pharmaceuticals based on the knowledge of their biological target. Such pharmaceuticals are typically, but not limited to, organic small molecules that either inhibit or activate the function of a target biological molecule. Typically, such a drug target is a key molecule involved in a particular metabolic or signalling pathway that is integral to a specific disease, condition or disorder or, relevant to the present invention, to the infectivity, survival and/or pathogenicity of a microbial pathogen. Non-limiting examples of biological molecules that may be the subject of drug design include enzymes, receptors and ion pumps.

Those skilled in the art would readily acknowledge that drug design involves, in its most basic sense, the design of small molecules that are complementary in shape and charge to the target region of a biological molecule with which they then interact and/or bind to.

Drug design commonly relies on, but is not limited to, either structure-based and/or computer-based modelling techniques.

Drugs may be designed that bind to the active region and/or active site of a target biological molecule and inhibit said molecule's functioning. Such inhibition may be sufficient to prevent, or at least partially inhibit, signalling of one or more pathways in which the target biological molecule functions. Furthermore, these drugs should also be designed so as to not target any "off-target" biological molecules that may be similar structurally to the target molecule as such off-target drug interactions may lead to undesirable side effects.

Inhibitors produced as a result of drug design may be organic small molecules produced through chemical synthesis or biopolymer-based drugs, so-called biologies, produced through biological processes. It should be understood, however, that this invention is not limited by reference to the specific methods of drug synthesis disclosed.

At the clinical level, screening a candidate inhibitor may include obtaining samples from test subjects before and after the subjects have been exposed to a test inhibitor. The levels in the samples of the protein product and/or activity of XAP may then be measured and analysed to determine whether the levels and/or activity of XAP change after exposure to the candidate inhibitor. By way of example, protein product levels in the samples may be determined by mass spectrometry, western blot, ELISA and/or by any other appropriate means known to one of skill in the art. Additionally, the activity of the protein products, such as their enzymatic activity, may be determined by any method known in the art. This may include, for example, enzymatic assays, such as spectrophotometric, fluorometric, calorimetric, chemiluminescent, light scattering, microscale thermophoresis, radiometric and chromatographic assays.

It would be appreciated that subjects who have been treated with a candidate inhibitor may be routinely examined for any physiological effects which may result from the treatment. In particular, the candidate inhibitors will be evaluated for their ability to treat and/or decrease the occurrence of a Mycoplasma infection in a subject.

A useful reference describing the general aspects, methods, and principles for drug screening, design and engineering are provided in Textbook of Drug Design and Development (4^th Edition, CRC Press F.L. USA, 2009) which is incorporated herein by reference.

In a related aspect, the invention provides an inhibitor of a XAP of a Mycoplasma spp. identified, designed and/or engineered by the method of the aforesaid aspect.

Suitably, the inhibitor is for use in the methods hereinbefore described.

In particular embodiments, one or more immunogenic fragments, one or more isolated proteins, one or more antibodies or antibody fragments and/or one or more inhibitors of XAP described herein may be included in a kit suitable for use in the methods of present invention. As such, the kit may further comprise, for example, additional diagnostic reagents such as secondary antibodies, enzymes (e.g., alkaline phosphatase or horseradish peroxidase), substrates for the enzymes (e.g., Luminol, ABTS or NBT), blocking agents and/or wash agents.

EXAMPLE 1

Materials and Methods

Detection and isolation of M. hyopneumoniae surface-expressed proteins. XAP was identified as being surface-expressed by trypsin shaving both freshly harvested Mhp cells and biotinylated Mhp cells. Methods used to biotinylate and recover surface proteins labelled with biotin and generate tryptic peptides of surface- exposed proteins (surface shaving) and characterise them by LC-MS/MS have been described previously (Bogema et al., J Biol Chem, 2011 ; Deutscher et al., J Proteome Res, 2012; Bogema et al., MBio, 2012). XAP was identified from separate (biological and technical replicates) shaving experiments, one of which was doubly biotinylated and shaved. Enzymatic cell surface shaving. Enzymatic cell surface shaving with trypsin was used to identify surface exposed proteins. Freshly harvested and washed Mhp cells were resuspended in PBS (pH 7.8) and pre-warmed with gentle mixing for 15 minutes at 37°C. A solution of 5 mg.mL^"1 cell culture grade trypsin [Sigma Aldrich] was pre-warmed along with the cells. A final concentration of 50 g.mL^"1 trypsin was added to the cells and allowed to incubate with gentle mixing for 5 minutes. After 5 minutes cells were immediately placed on ice and pelleted by centrifugation at 4000 ^χ g at 4°C. Supernatant containing liberated surface exposed proteins and peptides was removed and centrifuged to remove debris and any remaining intact cells at 10 000 ^χ g at 4°C for 20 minutes. Supernatant was pH corrected with 100 mM ammonium hydrogen carbonate (NH₄HCO₃) to pH >8 and reduced and alkylated with 5 mM tributylphosphine (TBP), 20 mM acrylamide monomers for 90 minutes at room temperature. For analysis by LC -MS/MS, sample was diluted with five volumes 100 mM NH₄HCO₃ and 1 μg Trypsin Gold [Promega] added and digested overnight at 37°C with gentle mixing. Sample was cleaned up using solid phase extraction (1 mL CI 8 HLB columns) before analysis by LC-MS/MS.

LC-MS MS is used to detect tryptic peptides released by shaving the surface of Mhp with the enzyme trypsin. Only proteins exposed on the cell surface should be detectable using this approach.

Cell surface biotinylation. Cell surface biotinylation was carried out on intact cells using Sulfo-NHS-LC-biotin, combined with avidin column purification and/or blotting to purify or identify biotinylated surface proteins. For surface biotinylation experiments, freshly harvested and washed Mhp cells were resuspended in PBS (pH 7.8) and biotinylated with 0.5 mg.mL^"1 EZ-Link Sulfo-NHS-LC-biotin (Thermo Scientific) for 30 s on ice. The reaction was then quenched with the addition of a final concentration of 50 mM Tris-HCl (pH 7.4) and incubated for 15 min. Cells were washed in three changes of PBS and pelleted by centrifugation at 4000 ^χ g for 10 minutes. Enzymatic cell surface shaving with trypsin was then performed as described above before analysis by LC-MS MS.

Immunofluorescence microscopy. Rabbit polyclonal antisera raised against XAP was first generated. To this end, XAP was cloned and recombinantly expressed in E. coli and subsequently purified by nickel affinity chromatography. The purified protein was then used to immunize a rabbit for the production of polyclonal antiserum. This antiserum was used to label fixed, non-permeabilized Mhp cells. These labelled Mhp cells were subsequently stained with goat anti-rabbit antibodies conjugated with Alexa Fluor 488 and analysed by conventional fluorescence microscopy.

Results

Trypsin shaving the surface of freshly harvested Mhp cells and subsequent LC-MS/MS revealed a subset of tryptic peptides that mapped to the XAP protein (MHJ_0659; UniProt Q4A929) (Figures 2-7). This was a surprising observation because XAP has not been previously described as surface expressed, but is rather predicted to reside intracellularly.

To further confirm this finding that XAP resides on the surface of Mhp cells, surface-accessible proteins were labelled with Sulfo-NHS-LC-biotin prior to surface shaving with trypsin and LC-MS/MS analysis. As demonstrated in Figure 8, these surface proteome studies also revealed tryptic peptides that mapped to XAP indicating that XAP indeed resides on the cell surface of Mhp.

Fluorescence microscopy of non-permeabilized Mhp cells probed with rabbit anti-XAP antiserum further confirms that XAP is indeed surface accessible with stained Mhp cells demonstrating a fluorescent ring of XAP staining surrounding the cell surface.

Therefore, ample data is provided to support a surface location for XAP, as determined by surface shaving, surface biotinylation, and immunofluorescence microscopy studies.

EXAMPLE 2

To ascertain the immunogenicity of XAP, overlapping peptides spanning XAP will first be created. These peptides will be coupled to a support, such as nitrocellulose or onto microtitre plates. These will then be separately exposed to convalescent swine antisera from different animals to detect linear epitopes of XAP that are recognized by antibodies raised naturally during infection of pigs with Mhp. In addition, the peptides will be separately exposed to antisera raised against swine that have been immunized with the commercial bacterin formulation that provides protection against Mhp. This will enable the mapping of suitable epitopes within XAP recognized by both infected (and/or sick) animals and infected protected animals.

EXAMPLE 3 Mass spectrometry experiments were performed to ascertain whether anti- XAP antibodies alter the efficiency of the enzymatic activity of XAP on Bradykinin. Four experiments were performed as listed below, each at pH 8.8 and in the presence of Co2+ ions. The anti-XAP described below was raised in rabbit against the whole (i.e. full length) protein of XAP, whose amino acid sequence is set forth in SEQ ID NO: 2.

1. Bradykinin alone

2. anti-XAP sera (luL) alone

3. XAP + Bradykinin (1 :20 wt/wt ratio)

4. XAP + Bradykinin (1 :20 wt/wt ratio) + anti-XAP sera (luL)

As can be observed in Figure 9, the 504 peak is decreased from 100% (Figure 9C) intensity to -20% intensity (Figure 9D) with the addition of anti-sera, indicating that the anti-XAP sera does indeed block the enzymatic activity of XAP on Bradykinin.

Discussion

XAP has been identified as a potential novel antigenic determinant of Mhp and Mpn, as surface expressed proteins are known play a central role in the interaction between pathogenic bacteria and their hosts. This protein is already known, as the Mhp and Mpn genomes have been sequenced previously, but XAP has never been described as surface expressed and is sometimes described as a "moonlighting protein". To date, however, there is no data available showing that this protein elicits an immune response in an animal, the nature of such an immune response or if the immune response is protective against Mhp or Mpn infection.

From Example 1, XAP (Xaa-Pro aminopeptidase, MHJ 0659, UniProt Number Q4A929) was identified as a surface accessible protein in Mhp and thus a potential antigenic determinant of Mhp md Mpn.

Of relevance, XAP is an aminopeptidase that is predicted to be important novel pathogenic determinant of Mhp and Mpn. Specifically, the XAP is likely to be a key molecule in the pathogenic armoury of Mhp as it is capable of cleaving both bradykinin (BK) and substance P (SP) both of which play an important role in regulating ciliary function. XAPs are highly specific, hydrolysing the peptide bond at penultimate proline residues on the N-terminus of the protein. Successful cleavage of SP or BK would release N-terminal arginine, and in the case of BK, potentially an additional proline residue. Furthermore, it is anticipated that XAP could also cleave and possibly inactivate the various molecules listed in Table 1 as they all share the XAP cleavage site which may have significant implications as to how Mhp and Mpn cause disease.

Whilst BK is rendered inactive by penultimate proline cleavage, the same cleavage event does not inactivate SP. It does, however, remove the protective conformation that the penultimate proline provides, leaving the peptide vulnerable to degradation by non- specific proteases, such as leucine aminopeptidases (LAPs). In this way, XAP may work in concert with other aminopeptidases to degrade SP and other biologically active peptides.

It is well known that BK and SP play a role in regulating cilial function and are critical to the maintenance of ciliary beating frequency. Clearly, the presence of an XAP on the cell surface of this pathogen would destroy the biological effectiveness of BK and potentially, substance P which effectively would disarm the mucociliary escalator, creating much more favourable conditions for Mhp and Mpn to flourish. Accordingly, the data disclosed hereinjndicate that XAP could potentially be a key vaccine component.

Additionally, it is likely that this protease is further capable of cleaving other important immunological effector molecules that play integral roles in the pathogenesis of Mhp and/or Mpn infection.

Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention.

All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference. Table 1. List of host molecules that display a XAP cleavag

Polypeptide N-terminal sequence

Bradykinin Arg-Pro-Pro-Gly-Phe...

Erythropoietin Ala-Pro-Pro-Arg-Leu...

Factor XII Ile-Pro-Pro-Trp-Glu...

Fibrin a-chain Gly-Pro-Arg-Val-Val...

Growth Phe-Pro-Ala-Met-Pro...

Hormone

IL 1β Ala-Pro-Val-Arg-Ser...

IL2 Ala-Pro-Thr-Ser-Ser...

IL6 Val-Pro-Pro-Gly-Glu...

IL10 Ser-Pro-Gly-Gln-Gly...

Neuropeptide Y Tyr-Pro- Ser-Lys-Pro ...

Peptide YY Tyr-Pro-Ile-Lys-Pro...

Plasminogen Glu-Pro-Leu- Asp-Asp...

Substance P Arg-Pro-Lys-Pro-Gln...

Claims

1. An immunogenic fragment of an isolated Xaa-Pro aminopeptidase of Mycoplasma.

2. The immunogenic fragment of Claim 1, wherein the isolated Xaa-Pro aminopeptidase comprises an amino acid sequence set forth in SEQ ID NO: 2.

3. The immunogenic fragment of Claim 1 or Claim 2, which comprises an active aminopeptidase site of the Xaa-Pro aminopeptidase.

4. The immunogenic fragment of any preceding claim, which comprises, consists or consists essentially of an amino acid sequence set forth in SEQ ID NO: 1.

5. The immunogenic fragment of any preceding claim, which comprises: (i) one or more metal binding residues of the Xaa-Pro aminopeptidase and/or one or more active site residues; or (ii) one or more deletions or substitutions of one or more metal binding residues and/or one or more deletions or substitutions of the aminopeptidase active site residues.

6. An isolated protein comprising one or a plurality of immunogenic fragments according to any one of Claims 1-5.

7. An isolated protein comprising an amino acid sequence of an Xaa-Pro aminopeptidase of Mycoplasma or an immunogenic fragment thereof according to any one of Claims 1-5, wherein the one or more metal binding residues of the isolated Xaa-Pro aminopeptidase or fragment thereof have been substituted or deleted and/or wherein one or more amino acids of the aminopeptidase active site have been substituted or deleted.

8. The isolated protein of Claim 6 or Claim 7, wherein the Xaa-Pro aminopeptidase comprises the amino acid sequence set forth in SEQ ID NO:2.

9. The isolated protein of Claim 8, wherein the immunogenic fragment of the Xaa-Pro aminopeptidase comprises the amino acid sequence set forth in SEQ ID NO: l .

10. The isolated protein of any one of Claims 6-9, wherein the one or more metal binding residues include at least one of: an aspartate (D) residue; a glutamate (E) residue; and a histidine (H) residue.

1 1. An isolated nucleic acid which comprises a nucleotide sequence that encodes, the immunogenic fragment of any one of Claims 1 to 5 or the isolated protein of any one of Claims 6 to 10, or which comprises a nucleotide sequence complementary thereto.

12. A genetic construct comprising: the isolated nucleic acid of Claim 11 operably linked or connected to one or more regulatory sequences in an expression vector.

13. A host cell transformed or transfected with a nucleic acid according to Claim 1 1 or the genetic construct of Claim 12.

14. A method of producing the isolated immunogenic fragment of any one of Claims 1 to 5 or the isolated protein of any one of Claims 6 to 10, comprising; (i) culturing the host cell of Claim 13; and (ii) isolating said immunogenic fragment or protein from said host cell cultured in step (i).

15. An antibody or antibody fragment which binds and/or is raised against an immunogenic fragment of any one of Claims 1 to 5 and/or the isolated protein of any one of Claims 6 to 10.

16. A composition for preventing or treating a Mycoplasma-associated disease, disorder or condition, comprising one or more immunogenic fragments of any one of Claims 1 to 5, one or more isolated proteins of any one of Claims 6 to 10, one or more isolated nucleic acids according to Claim 11, one or more genetic constructs according to Claim 12, one or more host cells according to Claim 13 and/or one or more antibodies or antibody fragments that bind or are raised against an Xaa- Pro aminopeptidase or are according to Claim 15, together with a pharmaceutically- acceptable diluent, carrier or excipient.

17. The composition of Claim 16, which is an immunogenic composition.

18. The composition of Claim 17 which is a vaccine.

19. A method of immunizing an animal, including the step of administering one or more immunogenic fragments of any one of Claims 1 to 5, one or more isolated proteins of any one of Claims 6 to 10, one or more isolated nucleic acids according to Claim 1 1, one or more genetic constructs according to Claim 12, one or more host cells according to Claim 13 and/or one or more antibodies or antibody fragments that bind or are raised against an Xaa-Pro aminopeptidase or are according to Claim 15 or the composition of any one of Claims 16 to 18 to said animal to thereby induce immunity to Mycoplasma in said animal.

20. A method of preventing or treating a Mycoplasma-associaled disease, disorder or condition in an animal, said method including the step of administering one or more immunogenic fragments of any one of Claims 1 to 5, one or more isolated proteins of any one of Claims 6 to 10, one or more isolated nucleic acids according to Claim 11, one or more genetic constructs according to Claim 12, one or more host cells according to Claim 13 and/or one or more antibodies or antibody fragments that bind or are raised against an Xaa-Pro aminopeptidase or are according to Claim 15 or the composition according to any one of Claims 16 to 18 to the animal to thereby prevent or treat the Mycoplasma-associated disease, disorder or condition in the animal.

21. A method of detecting Mycoplasma in a biological sample obtainable from an animal, said method including the step of detecting a cell surface-expressed Xaa-Pro aminopeptidase on one or more Mycoplasma cells in the biological sample.

22. The method of Claim 21, wherein the cell-surface expressed Xaa-Pro aminopeptidase is detected by an antibody or antibody fragment.

23. The method of Claim 21, wherein the antibody or antibody fragment; (i) binds or is raised against a substantially full length and/or wild-type Xaa-Pro aminopeptidase; or (ii) is according to Claim 15.

24. A method of detecting, inhibiting and/or preventing Mycoplasma growth and/or activity in vitro, including the step of applying an effective amount of an isolated protein of any one of Claims 7-10 and/or an antibody or antibody fragment that binds or is raised against an Xaa-Pro aminopeptidase or are according to Claim 15, to an in vitro substrate to thereby detect, inhibit and/or prevent Mycoplasma growth and/or activity in the substrate.

25. The method of Claim 24, the effective amount is a bactericidally and/or a bacteriostatically effective amount.

26. A method of identifying, designing and/or engineering of an inhibitor of a Xaa-Pro aminopeptidase of a Mycoplasma spp., said method including the steps of:

(ii) determining whether the candidate inhibitor at least partly reduces, eliminates, suppresses or inhibits an activity of the Xaa-Pro aminopeptidase.

27. The method of Claim 26, wherein the inhibitor is an antibody or a small organic molecule.

28. An inhibitor of a Xaa-Pro aminopeptidase of a Mycoplasma, identified, designed and/or engineered by the method of Claim 26 or Claim 27.