MYCOPLASMA SURFACE ENDOPROTEASE AND USES THEREOF

TECHNICAL FIELD THIS INVENTION relates to a Mycoplasma surface protein. More particularly, this invention relates to isolated immunogenic fragments of a surface endoprotease of Mycoplasma that may be useful in the prevention or treatment of a Mycoplasma- associated disease, disorder or condition and/or in the screening, designing and/or engineering of inhibitors of said surface protein.

BACKGROUND

Mycoplasma hyopneumoniae (Mhp) is the etiological agent of porcine enzootic pneumonia (PEP), a highly infectious and globally distributed swine respiratory disease. Symptoms of PEP include growth rate retardation, reduced feed conversion, and a greater susceptibility to secondary bacterial (7) and viral infections, including porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV) and porcine circovirus type 2 (PCV2) (2, 3). A number of broad-spectrum antibiotics are currently used to treat M. hyopneumoniae infections, but a greater number of antibiotics are used to prevent polymicrobial respiratory infections {4). Thus M. hyopneumoniae is a major contributor to antibiotic use in swine production. In 1991 , M. hyopneumoniae caused an estimated $ 1 billion economic loss in the USA alone (5). Recent estimates of loss due to this pathogen are in the order of $US10 per head. This estimate did not account for any environmental impact incurred by animal waste containing large quantities of multiple antibiotic resistant bacterial populations and significant quantities of antimicrobial residues (6).

Animal waste including pig effluent is used as organic fertilizer on agricultural lands, particularly in China (7). China is the world's largest producer of pork, generating at least five times the number of pigs produced in the USA, the second largest producer (7). Pork is the most heavily consumed meat and this trend is unlikely to change as the world's population grows towards nine billion by 2050. M. hyopneumoniae is endemic in pig herds globally and antibiotics are needed to curtain losses that arise due infection caused by this pathogen. Bacterin vaccines are used to control M. hyopneumoniae in conjunction with antibiotics; however, their efficacy is limited due to a minimal reduction in pathogen transmission and high production cost (8). Evidently, there is a pressing need to enhance our understanding of M. hyopneumoniae pathogenesis to develop more efficacious vaccines and therapeutics that seek to eradicate this pathogen by preventing colonization of the respiratory tract and reduce reliance on antibiotics.

A major innate barrier to all infectious respiratory microorganisms is the mucociliary escalator, which lines the respiratory tract and is composed of mucus secreting goblet cells and ciliated epithelium. Mucus traps inhaled particles that are then propelled to the pharynx by the synchronized beating of cilia to be either swallowed or expectorated (9). M. hyopneumoniae avoids mucociliary clearance by disrupting the mucociliary escalator via the initiation of ciliostasis, loss of cilial function and epithelial cell death. However, the mechanisms by which this is achieved are poorly understood (10). Cilioinhibitory factors deployed by other respiratory pathogens to disrupt the mucociliary system, such as the toxin pneumolysin of Streptococcus pneumoniae (11) or the low molecular weight glycopeptides produced by Haemophilus influenzae (12), have not been described for M. hyopneumoniae. The human respiratory pathogen, Mycoplasma pneumoniae, is believed to cause allergic-type inflammation by secreting the community-acquired respiratory distress syndrome (CARDS) toxin (13), though this toxin has not been found in M. hyopneumoniae.

However, a M. hyopneumoniae signal peptidase has recently been shown to be cytotoxic to mammalian cell (14), though this protease is not surface expressed (75). While mycoplasmas can cause some direct tissue damage through the production of the metabolic by-product hydrogen peroxide (16), this is not necessarily linked with pathology (17). For example, avirulent strains of M. pneumoniae do not have decreased levels of hydrogen peroxide (17), and mutants of Mycoplasma gallisepticum that are unable to produce hydrogen peroxide are virulent in a chicken model of pathogenesis (75).

Many bacterial species are known to manipulate host defenses to their advantage including actively attracting immune effector cells to the site of infection because host-derived proteases, released by neutrophils and macrophages, are more efficient at degrading extracellular matrix (ECM) components than bacterial-derived proteases (19). Host-induced ECM proteolysis represents a mechanism to source nutrients, a process that is vital for genome-reduced organisms unable to synthesize amino acids, nucleotides, fatty acids and other macromolecular building blocks (20). Mycoplasmas adhere to respiratory epithelium and elicit a number of defense mechanisms. These include the induction of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFa), interleukin (IL) 1 β, IL6, and 1 L8, stimulation of lymphocytes, and increase the cytotoxicity of macrophages and natural killer (NK) cells (21). Several other respiratory defense mechanisms are also up-regulated by non-self- recognition. Antimicrobial peptides (AMPs), such as lactoferrin, lysozyme, and cathelicidins, are secreted from respiratory epithelium and not only directly destroy pathogens but also act as effector molecules regulating both innate and adaptive immune systems (22). Inflammation is intensified by microbe-induced activation of enzymatic cascades, including the kallikrein/kinin system which releases bradykinin (BK), a potent bronchoconstrictor and pro-inflammatory peptide (23). Neurogenic inflammation also plays a major role in the innate immune response to infections, with non-myelinated C-fibres innervating the majority of the lung (24). These fibres secrete neuropeptides such as substance P (SP), neurokinin A (NKA), and neuropeptide Y (NPY) which are similar to AMPS in both structure and function (25), and are known to stimulate mucus secretion and increased ciliary beat frequency (CBF) (26, 27).

Despite fostering a formidable immune response, M. hyopneumoniae is associated with chronic illness, thus maintaining a balance of contrasting immunologic responses likely to impact virulence and disease progression. Host effector molecules and their receptors are susceptible to proteolytic modifications by bacterial proteases that render them either active or inactive (19). Despite evolving via a process of genome decay, M. hyopneumoniae has retained the genetic capacity to express several putative proteases, yet how these may affect their host has not been explored. For example, several mycoplasmas, including M. hyopneumoniae, have the capacity to inactivate BK, however the mechanism that underpins this activity was not determined (28).

PepF (sometimes referred to PepB) belongs to the M3B family of oligoendopeptidases that cleave at mainly hydrophobic residues at PI (Monnet et al, 1994) in peptides ranging between 8 and 17 amino acids in length (Nardi, Renault & Monnet, 1997). The M3B family also belongs to the Gluzincin superfamily, known to play important roles in disease pathologies (Seals & Courtneidge, 2003). PepF does not appear to be present in eukaryotes. However, it is found in some low G+C content Firmicutes, Spirochetes, Proteobacteria, Archaea and Protozoa (Kleine et al, 2008). Despite being relatively widespread among bacterial species, literature concerning PepF is relatively scarce compared to other Gluzincin family members such as angiotensin converting enzyme (ACE), neprilysin, and pseudolysin (Rawlings, Barrett & Bateman, 2012; Schomburg et al, 2013). SUMMARY

The present invention is predicated in part on the surprising discovery that an Oligoendopeptidase F (PepF) previously thought to be located intracellularly is present on the surface of Mycoplasma cells.

Accordingly, one form of the invention is broadly directed to fragments and/or mutants of a Mycoplasma PepF oligoendopeptidase and their use in preventing and/or treating a Mycoplasma-associated disease, disorder or condition.

In a first aspect, the invention provides a fragment of an isolated PepF protein of Mycoplasma, or a variant or derivative thereof.

Preferably, the isolated PepF protein comprises an amino acid sequence set forth in any one of SEQ ID NOS: 1 -5.

In one embodiment, the fragment comprises an active endopeptidase site of the isolated PepF protein. In another embodiment, the active endopeptidase site of the isolated PepF protein comprises one or more amino acid deletions and/or non- conservative substitutions.

In a particular embodiment, the fragment comprises one or more of residues 389H, 390E, 393H, 417E, 516H, 522Y and 526Y of SEQ ID NO: l .

In another particular embodiment, the fragment is a variant that has one or more of residues 389H, 390E, 393H, 417E, 516H, 522Y and 526Y of SEQ ID NO: l substituted or deleted.

In particular embodiments, the fragment is an immunogenic fragment.

This aspect also includes an isolated protein comprising one or a plurality of the aforementioned fragments. To this end, the isolated protein is suitably an immunogenic protein.

In a second aspect, the present invention provides an isolated nucleic acid that comprises a nucleotide sequence that encodes the fragment or the isolated protein of the first aspect or the variant or derivative thereof, or a nucleotide sequence complementary thereto.

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

In a fourth aspect, the invention provides a host cell transformed or transfected with an isolated nucleic acid of the second aspect or a genetic construct of the third aspect. In a fifth aspect, the invention provides a method of producing an isolated immunogenic fragment of the first aspect including; (i) culturing the transformed or transfected host cell of the fourth aspect; and (ii) isolating said fragment from said host cell cultured in step (i).

In a sixth aspect, the invention provides an antibody or antibody fragment which binds and/or is raised against an immunogenic fragment of the first aspect, or a variant or derivative thereof.

Suitably, said antibody or antibody fragment specifically binds said immunogenic fragment.

In one embodiment, the antibody is an inhibitory antibody. In a particular embodiment, the antibody blocks or inhibits an activity of PepF. In one embodiment, the antibody blocks or inhibits PepF cleavage of a neuropeptide and/or a proinflammatory peptide. The neuropeptide and/or proinflammatory peptide may be, or include, substance P (SP), bradykinin (BK) and/or neurokinin A (NKA).

In a seventh aspect, the invention provides a composition for preventing, treating or immunizing against aMycoplasma-associatod disease, disorder or condition, comprising: (i) an isolated PepF protein or one or more isolated fragments of the first aspect or a variant or derivative thereof; (ii) one or more isolated nucleic acids encoding a PepF protein or one or more isolated fragments of the first aspect or a variant or derivative thereof; (iii) one or more genetic constructs comprising the isolated nucleic acid of (ii); (iv) one or more host cells comprising the isolated nucleic acid of (ii) and/or the genetic construct of (iii); and/or (v) one or more antibodies or antibody fragments that bind or are raised against a Mycoplasma PepF protein, fragment, variant or derivative thereof; together with a pharmaceutically-acceptable diluent, carrier or excipient.

In an eighth aspect, the invention provides a method of eliciting an immune response in an animal including the step of administering to the animal: (i) an isolated PepF protein or one or more isolated fragments of the first aspect or a variant or derivative thereof; (ii) one or more isolated nucleic acids encoding a PepF protein or one or more isolated fragments of the first aspect or a variant or derivative thereof; (iii) one or more genetic constructs comprising the isolated nucleic acid of (ii); (iv) one or more host cells comprising the isolated nucleic acid of (ii) and/or the genetic construct of (iii); (v) one or more antibodies or antibody fragments that bind or are raised against a Mycoplasma PepF protein, fragment, variant or derivative thereof; and/or (vi) the composition of the seventh aspect; to thereby elicit an immune response to Mycoplasma in the animal.

In a ninth aspect, the invention provides a method of immunizing an animal including the step of administering to the animal: (i) an isolated PepF protein or one or more isolated fragments of the first aspect or a variant or derivative thereof; (ii) one or more isolated nucleic acids encoding a PepF protein or one or more isolated fragments of the first aspect or a variant or derivative thereof; (iii) one or more genetic constructs comprising the isolated nucleic acid of (ii); (iv) one or more host cells comprising the isolated nucleic acid of (ii) and/or the genetic construct of (iii); (v) one or more antibodies or antibody fragments that bind or are raised against a Mycoplasma PepF protein, fragment, variant or derivative thereof; and/or (vi) the composition of the seventh aspect to thereby induce immunity to Mycoplasma in the animal.

In a tenth aspect, the invention provides a method of treating or preventing a Mycoplasma-associated disease, disorder or condition, including the step of administering to the animal: (i) an isolated PepF protein or one or more isolated fragments of the first aspect or a variant or derivative thereof; (ii) one or more isolated nucleic acids encoding a PepF protein or one or more isolated fragments of the first aspect or a variant or derivative thereof; (iii) one or more genetic constructs comprising the isolated nucleic acid of (ii); (iv) one or more host cells comprising the isolated nucleic acid of (ii) and/or the genetic construct of (iii); (v) one or more antibodies or antibody fragments that bind or are raised against a Mycoplasma PepF protein, fragment, variant or derivative thereof; and/or (vi) the composition of the seventh aspect to thereby treat or prevent the disease, disorder or condition in the animal.

In an eleventh aspect, the invention provides a method of inhibiting or suppressing proteolysis of a neuropeptide and/or a proinflammatory peptide in an animal including the step of administering to the animal : (i) an isolated PepF protein or one or more isolated fragments of the first aspect or a variant or derivative thereof; (ii) one or more isolated nucleic acids encoding a PepF protein or one or more isolated fragments of the first aspect or a variant or derivative thereof; (iii) one or more genetic constructs comprising the isolated nucleic acid of (ii); (iv) one or more host cells comprising the isolated nucleic acid of (ii) and/or the genetic construct of (iii); (v) one or more antibodies or antibody fragments that bind or are raised against a Mycoplasma PepF protein, fragment, variant or derivative thereof; and/or (vi) the composition of the seventh aspect to inhibit or suppress proteolysis of the neuropeptide and/or proinflammatory peptide in the animal.

The neuropeptide and/or proinflammatory peptide may be, or include, substance P (SP), bradykinin (BK) and/or neurokinin A (NKA).

In a twelfth 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 PepF protein on one or more Mycoplasma cells in the biological sample.

In particular embodiments, the PepF protein is detected in the biological sample by binding an antibody or antibody fragment thereto.

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

(i) contacting a Mycoplasma PepF 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 the Mycoplasma PepF protein fragment, variant or derivative thereof.

In one embodiment, the activity of the PepF protein is endopeptidase activity, preferably towards a neuropeptide and/or a proinflammatory peptide. The neuropeptide and/or proinflammatory peptide may be, or include, substance P (SP), bradykinin (BK) and/or neurokinin A (NKA).

In another embodiment, the activity of the Mycoplasma PepF protein 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 Mycoplasma PepF protein identified, designed and/or engineered by the method of the thirteenth aspect.

In particular embodiments, the Mycoplasma is Mycoplasma hypopneumoniae, Mycoplasma pneumoniae, Mycoplasma bovis, Mycoplasma fermentans or Mycoplasma mycoides.

Suitably, the animal of the aforementioned aspects of the invention is a mammal.

Preferably, the mammal is a pig, human or bovine. BRIEF DESCRIPTION OF FIGURES

Figure 1. NCBI sequence viewer for Oligoendopeptidase F (MHJ_0522/AAZ44608.2). This diagram depicts an M3B peptidase family domain spanning amino acids 54-595, and active sites at 389H, 390E, 393H, 417E, 516H, 522 Y and 526Y (bold dictates zinc binding sites).

Figure 2. Phylogenetic tree showing evolutionary relationships of PepFs in relation to MHJ_0522. Tree calculated with phyml -d aa -q -n 1 -b -4 -m JTT -v e -c 4 -a e -s. Best Gblock options -bl=10 -b2=10 -b5=a Gblocks alignment: 572 positions (87 %) in 6 selected block(s). Alignment calculated with ClustalOmega.

Figure 3. Expressing Mhp PepF as a recombinant protein rMHJ_0522. A) SDS- PAGe gel showing purified rMHJ_0522 resolves as a single monomer of approximately 70 kDa. B) Tryptic peptide matches (bold and red) of rMHJ_0522 demonstrating 51% sequence coverage.

Figure 4. PepF is present on the surface of Mhp cells. A) Underlined: tryptic peptides released by mild trypsin digestion of cell surface proteins that map to MHJ_0522. Double underlined: tryptic peptides mapping to MHJ_0522 that were identified after biotinylated surface proteins were recovered by avidin chromatography and digested with trypsin. B) Images are illustrating surface localization of MHJ 0522. Whole Mhp cells were probed with rabbit anti-MHJ_0522 serum and anti-rabbit antibodies conjugated to Alexa Fluor 488 (red). Mhp cells were also stained with the nucleic acid stain DAP1 (blue).

Figure 5. rMHJ 0522 cleaves bradykinin (BK) at RPPGJ,F|SPFR. A) amino acid sequence of BK demonstrating two cleavage events and associated ion masses. B) An example of MALDI-MS/MS spectra demonstrating peaks for BK fragments after rMHJ_0522 was incubated with BK in the presence of cofactor Mn ²⁺ at pH 7.3 for one hr. The masses 652.74 Da, 572.66 Da, and 505.57 Da represent BK fragments BK5-9 (FSPFR), BK1-5 (RPPGF) and BK6-9 (SPFR) respectively. C) Control MALDI- MS/MS spectra of BK in the absence of rMHJ_0522. A single prominent peak observed at 1060.21 Da.

Figure 6. Cleavage of BK by rMHJ_0522 is influenced by pH and metal divalent cofactors. rMHJ 0522 produces fragments BK1-5, BK5-9, and BK6-9 at pH 6, 7.3 and 8.8 and in the presence of all cofactors tested. However, the intensity of each peak varies implying that at different pH levels and in the presence of various cofactors certain cleavages are more prevalent.

Figure 7. rMHJ_0522 cleaves SP at RPKPQQFF|G4L|M. A) Amino acid sequence of SP showing SP cleavage points by rMHJ_0522 and their corresponding mass (m/z). B) MALDI-TOF MS spectra of SP in the absence of rMHJ_0522 (single peak at SP intact mass 1347.7 Da). C) MALDI-TOF MS spectra of SP after incubated with rMHJ 0522 at pH 7.3 in the presence of Zn ²⁺ demonstrating additional peaks representative of the cleavage events shown in A). * denotes a common matrix contaminant at mass 379.0760.

Figure 8. Cleavage of SP by rMHJ_0522 is influenced by pH and metal divalent cofactors. At pH 6, only the cleavage fragment SP1-8 was produced with all cofactors tested. At pH 7.3, the most prevalent cleavage fragment was SP1-8. However the presence of Zn ²⁺ produced additional cleavage fragments SPl -9 and SPl-10, though at lower intensities. At pH 8.8 only SP1 -8 was produced. However the intensities for Co ²⁺ and Mn ²⁺ were lower by -50%.

Figure 9. rMHJ 0522 cleave NKA at HKTDSF|V jG^LM. A) Amino acid sequence of NKA is illustrating NKA cleavage events by rMHJ_0522 and their corresponding mass (m/z). B) Control MALDI-TOF MS spectra of NKA in the absence of rMHJ_0522 (single peak at NKA intact mass 1334.31 Da). C) An example of MALDI-TOF MS spectra of NKA after incubation with rMHJ_0522 at pH 7.3 in the presence of Zn ²⁺ . Additional peaks are representative of cleavage events shown in A).

Figure 10. Cleavage of NKA by rMHJ_0522 is influenced by pH and metal divalent cofactors. At pH 6.3 rMHJ_0522 only produced fragments in the presence of Co ²⁺ and Ca ²⁺ . At pH 7.3 rMHJ_0522 only produced all fragments in the presence of Zn ²⁺ with NKA1 -8 being the most prominent.

Figure 11. BLAST analysis of Mycoplasma PepF protein sequences. PepF amino acid sequences are as follows: SEQ ID NO: l = Mycoplasma hypopneumoniae; SEQ ID NO:2 = Mycoplasma pneumoniae; SEQ ID NO:3 = Mycoplasma bovis; SEQ ID NO:4 = Mycoplasma mycoides; and SEQ ID NO:5 = Mycoplasma fermentans. Figure 12. Amino acid sequence alignment for PepFs. Amino acid sequence alignment for PepFs Q4A9G3 (M. hyopneumoniae), E4PZ74 (M. bovis), C4XE87 (M. fermentans), PEPF_MYCPN (M. pneumoniae). All active sites are conserved across aligned species. Blue arrows denote active sites; Red box encapsulates the H-E-X-X-H (SEQ ID NO:6) motif of zinc metalloendopeptidases belonging to the MA protease clan. Figure 13. PepF and XAP cleavage of bradykinin. MHJ 0522 (PepF) (blue and green arrows) and MHJ 0659 (PepP/Xaa-Pro) (red arrow) combined cleavage of Bradykinin (BK) at pH 7.3 in the presence of Zn ²⁺ and Co ²⁺ . The green arrow at peaks at 573 Da and 506 Da are products of a single cleavage event; 573 Da represents the mass of BK1-5 (RPPGF), and 506 Da represents the mass of BK6-9 (SPFR), indicating the cleavage event occurs at RPPGF SPFR.

Figure 14. PepF and XAP cleavage of substance P. MHJ 0522 (PepF) (blue arrows) and MHJ_0659 (PepP/Xaa-Pro) (red arrow) combined cleavage of Substance P (SP) at pH 7.3 in the presence of Zn ²⁺ and Co ²⁺

DETAILED DESCRIPTION

The present invention arises, in part, from the identification of surface accessible PepF protein of Mycoplasma hyopneumoniae {Mhp), the main causative agent of PEP and a major pathogen of swine worldwide inflicting losses of billions of dollars per annum. Relevantly, the PepF protein is not traditionally thought to be surface exposed on Mhp, and thus the suitability of these proteins as immunogens and vaccine candidates is not foreseen. Furthermore, the PepF protein has endopeptidase activity towards neuropeptides such as substance P, which is a neuropeptide that induces an extensive innate immune response and facilitates mucociliary clearance, but only when bound to NKl and NK2 receptors. The C-terminal sequence G-L-M-NH2 is critical for NKl and NK2 receptor binding in pigs. The invention therefore provides methods for immunization and/or eliciting an immune response to PepF, by administering a PepF protein or fragment thereofand/or an inhibitory antibody or antibody fragment that binds PepF. Also provided are methods for inhibiting or suppressing proteolysis of a neuropeptide and/or a proinflammatory peptide by administering, for example, an inhibitory, mutated PepF having reduced protease activity and/or methods that identify PepF inhibitors, such as small molecule inhibitors. It is further proposed that these features of PepF protein of Mhp may also be relevant to PepF orthologues in other Mycoplasmas such as Mycoplasma pneumoniae, Mycoplasma bovis, Mycoplasma fermentans and/or Mycoplasma mycoides.

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 'W 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. hyopneumoniae, M. pneumoniae, M. bovis, M. fermentans, M. mycoides, M. orale, M. hominis, M. pulmonis, M. alvi, M. sualvi, M. iowae, M. moatsii, M. pirum, M. buccale, M. spermatophilum, M. salvarium, M. hominis, M. penetrans, M. hyorhinis, M. muris, M. fastidiosum, M. amphoriforme, M. genitalium, M. imitans, M. testudinis M. arthritidis and Ureaplasma urealyticum. In certain preferred embodiments, Mycoplasma or Mycoplasma spp. includes M. hyopneumoniae, M. pneumoniae, M. bovis, M. fermentans and M. mycoides, including and encompassing all serotypes and strains of these species.

Particular embodiments of isolated Mycoplasma PepF proteins comprise amino acid sequences set forth in SEQ ID NOS: l -5, shown in FIGS 3, 4 and 1 1 .

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. Isolated material may also, or alternatively, be in enriched, partially purified or purified 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. Suitably, the Mycoplasma PepF proteins such as according to SEQ ID NOS: 1- 5 are proteases, or more particularly endopeptidases, and even more particularly oligoendopeptidases .

As used herein, a "protease" is a protein which displays, or is capable of displaying, an ability to hydrolyse or otherwise cleave a peptide bond. A "peptidase ' is a protease that displays protease activity toward peptide substrates. An "endopeptidase ', inclusive of oligoendopeptidases, can cleave peptide bonds between amino acids that are not at the N- or C-terminus of the peptide.

In some aspects, the invention provides fragments of the isolated Mycoplasma PepF proteins, such as according to SEQ ID NOS: 1-5 or a variant or derivative thereof.

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, 300, 350, 400, 450, 550 or up to about 600 amino acids of an amino acid sequence.

Particular embodiments of the invention provide an immunogenic fragment of a Mycoplasma PepF protein. By "immunogenic" is meant capable of eliciting an immune response upon administration to an animal such as a pig. The immune response may include the production, activation or stimulation of the innate and/or adaptive arms of the immune system inclusive of immune cells such as B and/or T lymphocytes, NK cells, granulocytes, macrophages and dendritic cells and/or molecules such as antibodies, cytokines and chemokines, although without limitation thereto.

In a particular embodiment, the immunogenic fragment comprises, consists or consists essentially of an amino acid sequence set forth in any one of SEQ ID NOS: 1- 5, which essentially comprises the endopeptidase active site of the PepF.

Referring to SEQ ID NO: l , the PepF protein disclosed herein comprises an endopeptidase site comprising residues 389H, 390E, 393H, 41 7E, 516H, 522Y and 526Y. Residues 389H, 393H and 417E are metal (e.g. divalent metal cations such as zincy ⁾ binding residues. Orthologous active site residues in SEQ ID NOS: 2, 3 and 5 are shown in FIG. 12. Thus, the immunogenic fragment may comprise the entire endopeptidase active site of any one of SEQ ID NOS: 1 -5, or the immunogenic fragment may comprise a fragment of this active site sequence that comprises at least one of the active site residues thereof (e.g., one or more of residues 389H, 390E, 393H, 417E, 516H, 522Y and 526Y). The invention also provides an isolated protein comprising one or a plurality of fragments of a Mycoplasma PepF protein.

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 "polytope" 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 embodiment, the invention resides in an isolated protein comprising an amino acid sequence of a Mycoplasma PepF protein, variant, fragment or derivative thereof, and comprising at least one amino acid substitution or deletion of at least one of the active site residues of the PepF protein (e.g., one or more of residues 389H, 390E, 393H, 417E, 516H, 522Y and 526Y).

In yet a further embodiment, the invention of the present aspect provides an isolated protein or peptide that consists of: (i) a segment, domain, portion or region of one or more of the isolated Mycoplasma PepF proteins described herein, such as those according to SEQ ID NOS: 1-5, and inclusive of variants or derivatives thereof; and (ii) optionally one or more additional amino acid sequences. In this regard, the additional amino acid sequences are preferably heterologous amino acid sequences that can be at the N- and/or C-termini of the recited amino acid sequence of the PepF fragment, although without limitation thereto.

It is proposed that in particular embodiments, the metal binding residues of a PepF protein may play a pivotal role in the enzyme's activity as this protease cleaves peptide bonds through a cleavage event mediated by a water molecule that is activated by a divalent metal cation.

Accordingly, the isolated protein of this aspect, or a variant, fragment or derivative thereof, may act as an inhibitory mutant (such as a dominant negative mutant) of a PepF protein. As used herein, the term "inhibitory or dominant negative mutant" refers to a mutant PepF protein or fragment thereof, which lacks protease activity and when expressed or present intracellularly or extracellularly, competes with the endogenous protease for substrates, metal ions etc., and thereby at least partly inhibits or suppresses the protease activity of the endogenous PepF protein. Further to the above, it will be appreciated that such inhibitory or dominant negative mutant PepF proteins may also be immunogenic.

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 25,%, 30%, 35%, 40%, 45%, 50% or more preferably at least 55%, 60%o 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 any one of SEQ ID NOS: l-5.

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. It will be appreciated that SEQ ID NOS: l -5 may be considered as embodiments of Mycoplasma PepF orthologs.

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. (John 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' ^' ' 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 PepF protein 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 Mycoplasma hyopneumoniae (Mhp), published for example in Minion et al, J Bacteriol, Nov 2004; 186(21):7123-7133 (GenBank Accession No. AE017332), or Mycoplasma pneumoniae {Mpri), 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 acid' 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, methyl inosine, methyl adenosine 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 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%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity with an isolated nucleic acid of the invention.

In one particular embodiment, the isolated nucleic acid of the present aspect consists of: (i) a nucleic acid that encodes a segment, domain, portion or region of an isolated Mycoplasma PepF proteins described herein, such as those according to SEQ ID NOS: l-5, and inclusive of variants or derivatives thereof; and (ii) optionally one or more additional nucleic acid sequences. In this regard, the additional nucleic acid sequences are preferably heterologous nucleic acid sequences that can be at the 5' (5- prime) and/or 3' (3-prime) ends of the isolated nucleic acid sequence, although without limitation thereto.

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 methyl cytosine) 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 vector" 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. SJ9, 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.

As generally used herein, an "antibody" is or comprises an immunoglobulin protein. The term "immunoglobulin" includes any antigen-binding protein product of a mammalian immunoglobulin gene complex, including immunoglobulin isotypes IgA, IgD, IgM, IgG and IgE and antigen-binding fragments thereof. Included in the term "immunoglobulin" are immunoglobulins that are chimeric or humanised or otherwise comprise altered or variant amino acid residues, sequences and/or glycosylation, whether naturally occurring or produced by human intervention (e.g. by recombinant DNA technology).

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 endopeptidase activity of a PepF protein and/or may inhibit reduce, eliminate, inhibit or suppress binding of a PepF to metal ions and/or one or more substrate molecules.

In a particular embodiment, the antibody or antibody fragment may be administered to an animal to provide "passive immunity" to a Mycoplasma-associated disease, disorder or condition in the animal. In a particular embodiment, the antibody or antibody fragment may inhibit or suppress the protease activity of an endogenous Mycoplasma PepF protein. Suitably, the antibody is a neutralizing antibody.

In yet another embodiment, antibodies or antibody fragments may be used to detect cell surface-expressed PepF oligoendopeptidase.

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.

Antibodies 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 particular aspects, the invention provides compositions and/or methods of preventing, treating and/or immunizing against a Mycoplasma-associatQd 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 M/w)-associated disease, disorder or condition after it has begun to develop. The term "ameliorating," in respect of a Mycoplasma-assotiated 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 initiated prior to the onset of a symptom, aspect, or characteristic of a Mycoplasma-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 -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 Mycoplasma -associated disease, disorder or condition.

In the context of the present invention, by "Mycoplasma-associated disease, disorder or condition " is meant any clinical pathology resulting from infection by a Mycoplasma spp., such as those hereinafter described and inclusive of Mhp and Mpn.

Typically, Mhp and Mpn colonize 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 pneumoniae, and/or bronchitis in humans. Diseases and/or clinical symptoms associated with Mpn infection include pharyngitis, bronchitis, tonsillitis, pneumonia, septicaemia, haemolytic anaemia, rheumatoid arthritis, Stevens- Johnson syndrome, encephalitis, Guillain-Barre syndrome and fever, although without limitation thereto.

M. bovis is a pathogen of cows and other bovine animals. It can cause mastitis, arthritis and pneumonia and is implicated in the pathogenesis or exacerbation of bovine respiratory disease (BRD), also called "shipping fever".

M. mycoides is a pathogen of ruminant animals. It is known as the causative agent of contagious bovine pleuropneumonia (CBPP), a contagious lung disease of cattle.

A composition for preventing or treating a Mycoplasma-associated disease, disorder or condition may comprise: (i) an isolated Mycoplasma PepF protein or one or more isolated fragments of the first aspect or a variant or derivative thereof; (ii) one or more isolated nucleic acids encoding a PepF protein or one or more isolated fragments of the first aspect or a variant or derivative thereof; (iii) one or more genetic constructs comprising the isolated nucleic acid of (ii); (iv) one or more host cells comprising the isolated nucleic acid of (ii) and/or the genetic construct of (iii); and/or (v) one or more antibodies or antibody fragments of the sixth aspect; together with a pharmaceutically- acceptable diluent, carrier or excipient.

By "pharmaceutically-acceptable carrier, diluent or excipienf 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.

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 excipient".

Useful carriers are well known in the art and include for example: thyroglobulin; albumins such as human serum albumin; toxins, toxoids or any mutant cross-reactive material (CRM) of the toxin from tetanus, diphtheria, pertussis, Pseudomonas, E. coli, Staphylococcus, and Streptococcus; polyamino acids such as poly(lysine:glutamic acid); influenza; Rotavirus VP6, Parvovirus VP1 and VP2; hepatitis B virus core protein; hepatitis B virus recombinant vaccine and the like. Alternatively, a fragment or epitope of a carrier protein or other immunogenic protein may be used. For example, a 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 excipient 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-derived adjuvants such as Corynebacteriwn parvum; Propionibacterium-dQnvQd 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, N,N-dicoctadecyl-N', N'bis(2- hydroxyethyl-propanediamine), methoxyhexadecylglycerol, and pluronic polyols; polyamines such as pyran, dextransulfate, poly IC carbopol; peptides such as muramyl dipeptide and derivatives, 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 ISCOMATRIX® 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 Carbopol' 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.

Compositions and vaccines of the invention may be administered 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 al , 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 N.Y. 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 PepF fragment, protein, immunogenic protein, encoding nucleic acid, antibody, antibody fragment or composition disclosed herein into an animal subject by a particular, chosen route.

Any safe route of administration may be employed, inclusive of oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intra-muscular, intra-dermal, transdermal, subcutaneous, inhalational, intraocular, intraperitoneal and intracerebroventri cul ar adm in i strati on . 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.

One particular broad application of the present invention is provision of methods of preventing, treating or immunizing an animal by administering to the animal (i) an isolated Mycoplasma PepF protein or one or more isolated fragments of the first aspect or a variant or derivative thereof; (ii) one or more isolated nucleic acids encoding a PepF protein or one or more isolated fragments of the first aspect or a variant or derivative thereof; (iii) one or more genetic constructs comprising the isolated nucleic acid of (ii); (iv) one or more host cells the isolated nucleic acid of (ii) and/or the genetic construct of (iii); (v) one or more antibodies or antibody fragments of the sixth aspect; and/or (vi) the composition of the seventh aspect.

The compositions and methods disclosed herein may elicit an immune response as hereinbefore described, providing at least partial immunity or protective immunity to a Mycoplasma.

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.

In this context "immunization" achieves or provides at least partial or temporary protection against a subsequent Mycoplasma infection. Immunization may be "active" through elicitation of a protective immune response or "passive" such as by administration of one or more antibodies or antibody fragments disclosed herein.

One particular embodiment relates to the finding that the M. hypopneumoniae PepF protein (SEQ ID NO: 1 ) has endopeptidase activity towards neuropeptides such as substance P, which is a neuropeptide that induces an extensive innate immune response and facilitates mucociliary clearance, but only when bound to NK1 and NK2 receptors. The C-terminal sequence G-L-M-N2 is critical for NK 1 and NK2 receptor binding in pigs. Thus, it is proposed that by blocking or inhibiting PepF oligoendopeptidase activity towards neuropeptides such as substance P, the innate immune response may be improved through facilitating binding of substance P to NK1 and NK2 receptors.

Thus, in a particular embodiment the elicitation of an antibody response in the animal, or the administration of antibodies or antibody fragment to the animal {i.e. by passive immunization), may enhance mucociliary clearance through facilitating binding of substance P to NK1 and NK2 receptors and thereby enhancing the innate immune response.

Accordingly, in one aspect the invention resides in a method of at least partly inhibiting or suppressing proteolysis of a neuropeptide and/or a proinflammatory peptide in an animal including the step of administering to the animal: (i) an isolated PepF protein or one or more isolated fragments described herein or a variant or derivative thereof; (ii) one or more isolated nucleic acids encoding a PepF protein or one or more isolated fragments described herein or a variant or derivative thereof; (iii) one or more genetic constructs described herein (iv) one or more host cells described herein; (v) one or more antibodies or antibody fragments that bind or are raised against a Mycoplasma PepF protein, fragment, variant or derivative thereof; and/or (vi) the composition described herein to at least partly inhibit or suppress proteolysis of the neuropeptide and/or proinflammatory peptide in the animal.

In particular embodiments, the isolated PepF protein comprises at least one amino acid substitution or deletion of at least one of the active site residues of the PepF protein (e.g., one or more of residues 389H, 390E, 393H, 417E, 516H, 522Y and 526Y), as hereinbefore described. In this regard, it is intended that the isolated PepF protein may act as an inhibitory or dominant negative mutant of a wildtype PepF protein.

Suitably, the method further includes the step of administering an XAP protein or an immunogenic fragment thereof that includes one or more metal binding residues; and/or a mutant XAP protein or fragment thereof that comprises one or more mutated metal binding residues, such as those described herein.

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 yet another embodiment the mammal is a bovine.

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.

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 an endogenous PepF 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 endogenous PepF protein is detected in the biological sample by an antibody or antibody fragment that binds to, or is raised against, a PepF protein, or an immunogenic fragment thereof. In certain other embodiments, the Mycoplasma PepF protein in the subject is detected in the biological sample by binding a small molecule thereto.

Suitably, detecting PepF includes the step of forming a detectable complex between an antibody, antibody fragment or the small molecule and the endogenous PepF protein. The complex so formed may be detected by any technique, assay or means known in the art, including immunoblotting, immunohistochemistry, immunocytochemistry, immunofluorescence, 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 PepF protein 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-PepF treatment, such as by a so- called "companion diagnostic".

In a further embodiment, 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 PepF protein, fragment or variant thereof and/or an antibody or antibody fragment to a substrate in vitro to thereby detect, inhibit and/or prevent Mycoplasma growth and/or activity in the substrate.

This may involve, but is not limited to, coating filters, plates and other cell culture equipment with an antibody and/or antibody fragment to PepF. 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.

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 PepF. 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 a PepF protein of Mycoplasma, said method including the steps of:

(i) contacting a Mycoplasma PepF 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 the Mycoplasma PepF protein, fragment, variant or derivative.

In some embodiments, the inhibitor may at least partly reduce, eliminate, inhibit or suppress the endopeptidase activity of the Mycoplasma PepF protein, fragment, variant or derivative.

In other embodiments, the inhibitor would act to at least partly reduce, eliminate, inhibit or suppress the ability of the Mycoplasma PepF protein, fragment, variant or derivative to bind to one or more other molecules or atoms, such as a substrate molecule or a 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 endopeptidase activity of a Mycoplasma PepF protein, fragment, variant or derivative 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 PepF protein may then be measured and analysed to determine whether the levels and/or activity of PepF 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- Edition, CRC Press F.L. USA, 2009) which is incorporated herein by reference.

In a related aspect, the invention provides an inhibitor of a Mycoplasma PepF protein, fragment, variant or derivative 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 a PepF protein 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.

In an embodiment, the invention contemplates one or more other Mycoplasma surface proteins, fragments thereof (inclusive of inhibitory protease-deficient mutants), antibodies thereto, encoding nucleic acids and/or compositions comprising same used in combination with the PepF protein, fragments thereof (inclusive of inhibitory protease-deficient mutants), antibodies thereto, encoding nucleic acids and/or compositions comprising these. In one particular embodiment, the one or more other Mycoplasma surface proteins include the Mycoplasma Xaa-Pro aminopeptidase (XAP) described in International Publication WO2015/123728. With particular regard to FIGS. 13 and 14, PepF and XAP in combination cleave bradykinin (BK) and substance P, in which case blocking or inhibiting this co-cleavage event may be particularly effective in preventing or treating diseases, disorders or conditions associated with Mycoplasma infection. By way of example, in some embodiments for the creation of an interfering mutant, one or more metal binding residues of XAP may by modified, such as at least one of: an aspartate (D) residue; a glutamate (E) residue; and a histidine (H) residue. In other embodiments, an immunogenic fragment comprising one or more of the metal binding residues of XAP may be used to elicit an immune response to XAP. For the XAP amino acid sequence set forth in SEQ ID NO:2 of WO201 5/123728, the residues are: 190H, 207D, 218D, 282H, and 324E.

Thus, combination therapy aimed at blocking or inhibition of both PepF and XAP may be particularly advantageous for preventing or treating Mycoplasma- associated diseases, disorders or conditions. So that the invention may be readily understood and put into practical effect, reference is made to the following non-limiting Examples.

EXAMPLES INTRODUCTION

We identified a putative surface-exposed Mhp oligoendopeptidase F (PepF; MHJ 0522) via three independent methodologies, despite this protein lacking evidence of a signal peptide, transmembrane domains, or stretches of hydrophobic amino acids sufficient to traverse the cell membrane. This protein was also predicted by PSORTb to reside within the cytosol of Mhp.

In the present example, we cloned and expressed recombinant MHJ 0522 (rMHJ_0522) as a polyhistidine fusion protein, and determined its activity against three substrates involved in mucociliary clearance: BK, NKA, and SP. Freshly cultured Mhp cells were run through SP affinity columns which two surface exposed moonlighting proteins, suggesting SP can be retained on Mhp surface. The expressed and purified recombinant MHJ 0522 hexa histidine fusion protein (rMHJ_0522) has the capacity to completely degrade the C-terminal sequence G-L-M-NH2 of Substance P, a neuropeptide, secreted in the lungs and from host effector molecules, that induces an extensive innate immune response and facilitates mucociliary clearance, but only when bound to NK1 and NK2 receptors. The C-terminal sequence G-L-M-NH2 is critical for NK1 and NK2 receptor binding in pigs. rMHJ_0522 cleavage of Substance P removes its ability to mediate inflammation. Additionally, substance P belongs to a superfamily of neuropeptides that all possess the same C- terminal sequence, including neurokinin A which, together with substance P, causes neurogenic inflammation in the lungs. As MHJ 0522 modulates the porcine innate immune system to the pathogens advantage, by targeting MHJ 0522 an infected pig would have greater success in clearing M. hyopneumoniae. It also makes an attractive therapeutic target as PepF is a prokaryotic protein and not found in eukaryotes.

METHODS AND MATERIALS

Mycoplasma hyopenumoniae culture conditions. M. hyopneumoniae strain J was cultured in Friss medium for 48 h at 37 ^° C and harvested by centrifugation at 12 000 x g for 15 min. Pellets were stored at -80 ^° C until use.

Detection and isolation of Mhp surface-expressed proteins. PepF was identified as being surface-expressed by immunofluorescence microscopy and 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, 201 1 ; 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

-1

15 minutes at 37°C. A solution of 5 mg.mL cell culture grade trypsin [Sigma

- 1

Aldrich] was pre-warmed along with the cells. A final concentration of 50 μg.mL 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 x g at 4°C. The supernatant containing liberated surface exposed proteins and peptides were removed and centrifuged to remove debris and any remaining intact cells at 10 000 ^χ g at 4°C for 20 minutes. The supernatant was pH corrected with 100 mM ammonium hydrogen carbonate (NH4HC03) to pH >8 and reduced and alkylated with five mM tributylphosphine (TBP), 20 mM acrylamide monomers for 90 minutes at room temperature. For analysis by LC- MS/MS, the sample was diluted with five volumes 100 mM NH4HC03 and one μg Trypsin Gold [Promega] added and digested overnight at 37°C with gentle mixing. The sample was cleaned up using solid phase extraction (1 mL C18 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 i n PBS (pH

7.8) and biotinylated with 0.5 mg.mL ^" ^ 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. Polyclonal antibodies against rMHJ_0522 were first raised in rabbits by the Institute and Medical and Veterinary Science [Australia], Microscopy was then performed following the same protocol as in (Robinson et al, 2013). Briefly, 1 ml of Mhp strain J culture was centrifuged at ten 000xg for 10 min and washed three times with 1 ml sterile PBS. A 1 in 100 dilutions of cells was made in PBS and added to glass coverslips and allowed to settle for 15 min at room temperature. Paraformaldehyde (4%) was added and incubated at room temperature for 30 min. Non-specific binding sites were blocked using 2% BSA in PBS overnight at 4°C. Cells were incubated with either a 1 in 100 dilution of rMHJ_0522 antisera or control rabbit sera for one h at room temperature, followed by one h incubation at room temperature with 1 in 1000 dilution of goat anti-rabbit antibodies conjugated to Alexa Fluor 488 [Life Technologies]. Control sera were collected from rabbits before immunization with rMHJ_0522. Coverslips were mounted in VECTASHIELD onto microscope slides and imaged using an Olympus BX51 Upright Epi Fluorescence microscope. Images were captured using an Olympus DP97 Digital Microscope Camera coupled with Olympus DP Controller software.

Expression and purification of rMHJ 0522. The mhj_0522 gene encoding PepF was ligated into expression vector PS 100030, conveying both a hexa histidine tag and ampicillin resistance, by Blue Heron Biotech (USA). All in frame TGA codons were substituted to TGG. The recombinant construct was transformed into Escherichia coli BL21 [Invitrogen] as per the manufacturer instructions, and grown overnight in LB supplemented with 100 mg/mL ampicillin. rMHJ_0659 was then purified under native conditions using 50% slurry of Profinity immobilized metal affinity chromatography Ni2+ charged resin [Bio-Rad], as per manufacturer's instructions. Once rMHJ_0659 was eluted using imidazole, it was dialysed against PBS in 10 K MWCO dialysis tubing stored at 4°C. ID SDS-PAGE was used to separate purified protein samples.

Proteomics. To analyses, purified protein samples separated by ID SDS-PAGE, peptide preparation, and MS analysis parameters were followed as described previously (Bogema et al, 2011). A novel assay was used to determine substrate cleavage. From a stock solution (1 mg/ml) of BK or SP [Sigma Aldrich] Ι μΐ was diluted in 8.5 \iL 50 mM Tris-HCl buffer (pH 7.5) and 0.5 \\L 100 mM divalent cation cofactor. Purified rMHJ_0522 was added in a 1 :20 protease to substrate concentration and incubated for 60 min at 37°C. The peptides were then desalted and captured using C18 ZipTips [Millipore]. Ι μΐ of peptide sample was then spotted onto a clean 384-well OptiTOF target plate [AB Sciex] followed by 1 μΐ of 5 mg/ml a-Cyano-4-hydroxycinnamic acid (CHCA) dissolved in 50% ACN, 0.1% TFA, ten mM NH4H2P04 and allowed to dry. Spotted samples were then analysed using a 5800 MALDI-TOF/TOF MS in positive reflector mode. Laser intensity was set to 2600 for MS parent ion scans and 3000 for MS/MS fragmentation ion scans. 400 laser shots were averaged for MS scans ad up to 1250 shots were averaged for MS/MS scan with the Dynamic Exit algorithm selected, which monitors spectral quality and stops shot accumulation if a user defined threshold is met. MS parent ion scans were calibrated using the fragments of Glu-Fibrinopeptide B present in the TOF/TOF standards mixture. The resulting MS spectral data was then manually inspected to explain the ions present concerning their amino acid sequence and the cleavage events caused by PepF proteolysis. Peaks found in both control and experiment data not pertaining to the parent molecule (artefacts from matrix) were removed for clarity.

Bioinformatics. Intracellular localization and presence of TMDs were predicted by PSORTb and TMpred respectively. The presence of conserved domains and active sites were ascertained using both NCBI sequence viewer, and MyHits. The phyletic tree was produced using the HOGENOM database.

RESULTS

Identification and cell localisation of MHJ 0522 in Mhp. The mhj_0522 gene of Mhp is annotated to encode for a putative PepF (MHJ 0522). While this protein possesses a M3B domain, and zinc protease signature (Fig. 1) indicating function as an oligoendopeptidase F, it has low sequence identity to other bacterial PepFs with the highest sequence identity to a non-mycoplasmal species reaching only 32% to PepB of Bacillus endophytics (BEH_03885). A phylogenetic tree indicates this PepF is unique to mycoplasmas (Fig. 2).

MHJ_0522 had a predicted MW of 71 kDa and indeed rMHJ_0522 resolved during SDS-PAGE as a single band within the correct range (Fig. 3a). This band was digested with trypsin and confirmed to be rMHJ_0522 by LC-MS/MS with 51% sequence coverage (Fig 3b). PepF is predicted to reside intracellularly yet surprisingly after trypsin shaving the surface of freshly harvested Mhp cells and subsequent LC-MS/MS a subset of tryptic peptides that mapped to the PepF protein were revealed (Fig. 4a). When freshly cultured M. hyopneumoniae cells were labeled with biotin and surface-exposed biotinylated proteins and subsequently recovered using avidin chromatography, LC-MS/MS identified more peptides mapping to MHJ_0522 (Fig. 4a).

To confirm the surface localization of PepF fluorescence microscopy of non- permeabilized Mhp cells were probed. Bound mono-specific polyclonal antibodies raised against rMHJ_0522 were detected on the surface of freshly cultured Mhp using anti-rabbit antibodies conjugated with Alexa Fluor 488 (Fig. 4b). rMHJ 0522 cleaves bradykinin (BK). Full-length BK [RPPGFSPFR] has a molecular mass of 1060.21 Da. In the absence of rMHJ_0522, a single prominent peak at 1060.21 Da was observed in MALDI/TOF/MS spectra (Fig. 5C). Once BK was incubated with rMHJ_0522 three additional peaks were observed, 652.74 Da, 572.66 Da, and 505.57 Da, demonstrating two cleavages of BK by rMHJ_0522 (Fig. 5A &5B). The peaks at 572.66 Da and 505.57 Da represent the same cleavage event; 572.66 Da represents the mass of BK1-5 (RPPGF), and 505.57 Da represents the mass of BK6-9 (SPFR), meaning the cleavage event occurs at RPPGF|SPFR. The peak at 652.74 Da equates to the mass of BK5-9 (FSPFR) thus represents another cleavage event at RPPGjFSPFR. rMHJ_0522 was also incubated with BK1-7 and was found to be ineffective in cleaving this BK fragment (data not shown) which consolidates rMHJ_0522 function as an oligoendopeptidase (i.e. inability to cleave peptides shorter than eight amino acids in length). The effect of divalent cofactors Ca ²⁺ , Co ²⁺ , Mn ²⁺ and Zn ²⁺ at pH levels 5, 6, 7.3 and 8.8 on rMHJ_0522 activity was tested. It was ascertained that rMHJ_0659 did not cleave BK at pH 5 (data not shown). At pH 6 Mn ²⁺ produced the most intense peak for BK5-9 and both Ca ²⁺ and Zn ²⁺ produced the most intense peaks for BK1-5. A similar profile was seen at pH 7.3; only Mn ²⁺ joined Ca ²⁺ and Zn ²⁺ in produced the most intense peak for BK1-5. In basic conditions, lower intensity peaks were observed for BK5-9, and Ca2+ and Co2+ produced the most intense peaks for BK1-5 (Fig. 6). rMHJ_0522 cleaves substance P (SP). Full-length SP [RPKPQQFFGLM] has a molecular mass of 1348 Da and control experiments demonstrated a single peak at this mass range. When SP was incubated with rMHJ_0522 additional peaks were observed at 1217 Da, 1 105 Da and 1048 Da corresponding to the mass of SP fragments SP1-8 [RPKPQQFF], SP1-9 [RPKPQQFFG], and SPl-10 [RPKPQQFFGL], respectively (Fig. 7). The most prevalent cleavage fragment generated across all pH (6, 7.3 & 8.8) and cofactors (Ca ²⁺ , Co ²⁺ , Mn ²⁺ , & Zn ²⁺ ) tested was SP1 -8 (Fig. 8), however at pH 7.3 in the presence of Zn ²⁺ , SP1 -9 and SP l -10 were also produced, albeit at lower intensities (Fig. 8). This data indicates that rMHJ_0522 can produce cleavage events at RPKPQQFF jGjLIM (red arrow indicates most prominent cleavage). Like BK, rMHJ_0522 did not cleave NKA at pH 5 (data not shown).

rMHJ_0522 cleaves neurokinin A (NKA). Full-length NKA [HKTDSFVGLM] has a molecular mass of 1 134 Da and control experiments demonstrated a single prominent peak at this mass range. When NKA was incubated with rMHJ_0522 additional peaks were observed at 890 Da, 833 Da and 734 Da corresponding to the mass of NKA fragments NKA 1-6 [HKTDSF], NKA1-7 [HKTDSFV], and NKA 1 -8 [HKTDSFVG], respectively (Fig. 9). At pH 6.3 only in the presence of Ca ²⁺ and Co ²⁺ did rMHJ_0522 produce NKA fragments, with NKA1 - 6 being the most prevalent in the presence of Ca ²⁺ , and NKA1-8 the most prominent in the presence of Co ²⁺ . At pH 7.3 only in the presence of Zn ²⁺ did rMHJ_0522 generate all NKA fragments, with NKA1 -8 being most prevalent. The other tested cofactors did not stimulate the cleavage needed to produce NKA1 -7 and Co ²⁺ only stimulated the production of NKA1-8. Like BK and SP, no rMHJ_0522 activity was observed at pH 5 (data not shown), but unlike BK and SP, no activity against NKA was observed at pH 8.8. rMHJ_0522 and XAP cleavage of bradykinin and substance P. As shown in FIG. 13, MHJ 0522 (PepF) (blue and green arrows) and MHJ 0659 (PepP/Xaa- Pro) (red arrow) combined to cleave bradykinin (BK) at pH 7.3 in the presence of Zn ²⁺ and Co ²⁺ . The green arrow at peaks at 573 Da and 506 Da are products of a single cleavage event; 573 Da represents the mass of BK1-5 (RPPGF), and 506 Da represents the mass of BK6-9 (SPFR), indicating the cleavage event occurs at RPPGF SPFR. The same protocol was followed as previously described with the following adjustments: both proteases, and both zinc and cobalt cofactors were added due to protease preference (PepF for zinc, PepP/Xaa for cobalt) Referring to FIG. 14, MHJ_0522 (PepF) (blue arrows) and MHJ_0659 (PepP/Xaa-Pro) (red arrow) combined to cleave substance P (SP) at pH 7.3 in the presence of Zn ²⁺ and Co ²⁺ . rMHJ 0659 and rMHJ 0522 share substrates and have the potential to cleave many biologically active proteins. rMHJ 0659 was shown to cleave at N- terminal penultimate prolines when the N-terminal amino acid was arginine (BK and SP), proline (BK second cleavage) and tyrosine (NPY). rMHJ_0522 cleaved BK at glycine and phenylalanine, SP at phenylalanine, glycine and leucine, and NKA at phenylalanine, valine, and glycine (Table 1). BK and SP were common substrates for both proteases. Interestingly, penultimate prolines are present at the N-terminal of many porcine innate system polypeptides, and the F-X-G-L-M-NH2 motif is common to all tachykinins, the largest family of neuropeptides (Table 2). We propose that these innate effector peptides and neuropeptides are potential substrates for PepP and PepF proteases on the surface of M hyopneumoniae.

Table 1 : Peptide substrates for rMHJ_0659 and rMHJ_0522. Blue arrows and red arrows represent rMHJ_0659 and rMHJ_0522 cleavage sites respectively.

Table 2: Porcine innate immune system polypeptides that exhibit penultimate proline (left) residues and tachykinin C-terminal sequences (right).

DISCUSSION

PepF has been identified as a potential novel antigenic determinant of Mhp 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 genome has been sequenced previously, but PepF was predicted to reside intracellularly. We have comprehensively demonstrated through three independent methodologies that PepF is found on the surface of Mhp.

Of relevance, PepF is a protease that is predicted to be an important novel pathogenic determinant of Mhp, and belongs to a super family of proteins associated with pathologies. Specifically, PepF is likely to be a key molecule in the pathogenic armory of Mhp as it is capable of cleaving and inactivating bradykinin (BK) neurokinin A (NKA), and substance P (SP) all of which play important roles in regulating a host's innate immune response to invading pathogens.

It is well known that BK, SP, and NKA play a role in regulating ciliary function and are critical to the maintenance of ciliary beating frequency (CBF). Clearly, the presence of a PepF on the cell surface of this pathogen would destroy the biological effectiveness of three innate immunity molecules which effectively could disarm the mucociliary escalator, creating much more favourable conditions for Mhp to flourish. Accordingly, the data disclosed herein indicate that PepF could potentially be a key vaccine component.

We have expressed and purified a recombinant hexa histidine fusion rMHJ_0522 and discovered to possess the ability to completely destroy the receptor binding capacity of several important host immune molecules and thus may be involved in disease pathogenesis. Therefore, elimination or reduction of this activity by generating an immune response to the active site or the metal -binding regions by the administration of inhibitors of this activity (e.g., monoclonal antibodies, which we have generated) may have efficacy in treating or preventing Mycoplasma- associated diseases, disorders or conditions. Relevantly, PepF is not traditionally thought to be surface exposed on Mhp and thus the suitability of these proteins as immunogens and vaccine candidates is not foreseen and are therefore novel. Additionally, PepF is only found in prokaryotes so cross-reactivity in eukaryotes, including pigs, is highly unlikely.

Previously vaccines candidates against M p have focused on the major adhesin proteins which bind directly to heparin, fibronectin, and plasminogen thus facilitating attachment to and colonisation of respiratory cilia. However, these candidates have had less success than the commercially available bacterin vaccines. PepF belongs to a different class of proteins called proteases. Likened to molecular knives, proteases are enzymes that cleave peptide bonds in polypeptide chains and profoundly influence protein shape, size, composition, localisation, turnover, and degradation. The effects of such influence include gain, loss or switch in protein functions. Proteases are thereby responsible for a multitude of physiological processes in all organisms, but they also play prominent roles in pathogenesis and are recognized as important virulence factors. Pathogens indisputably utilise proteolysis to establish successful infections, and this has driven research into developing protease inhibitors targeting their catalytic sites as therapeutic agents. Efficacious examples include human immunodeficiency virus aspartyl protease inhibitors, which are used to prevent the progression of acquired immunodeficiency syndrome, and two serine protease inhibitors used for the treatment of chronic hepatitis C infections.

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. REFERENCES

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