AU716700B2 - Mycoplasma recombinant polypeptides and vaccines - Google Patents

Description

SWO 97/21727 PCT/AU96/00803 1 MYCOPLASMA RECOMBINANT POLYPEPTIDES AND VACCINES The present invention relates generally to peptides and polypeptides and their use in vaccine preparations. More particularly, the present invention is directed to a peptide or polypeptide or a derivative, homologue or analogue thereof which corresponds to, mimics, or cross-reacts with, B-cell or T-cell epitopes on polypeptides encoded by Mycoplasma pneumoniae and M. genitalium. Vaccine preparations comprising the peptides or polypeptides of the present invention are useful in protecting individuals against infections by species of the genus Mycoplasma.

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

Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.

Sequence identity numbers (SEQ ID Nos.) for the nucleotide and amino acid sequences referred to in the specification are defined after the bibliography.

The micro-organism Mycoplasma pneumoniae is a pathogen of humans that typically colonises the upper respiratory tract. Mycoplasmapneumoniae moves along the cilia of the respiratory epithelium until in close association with the host cell to which it adheres. It disrupts the protein ciliary necklace at the base of the host cell cilia causing ciliostasis.

The threat that M. pneumoniae poses to children and sensitized adults is considerable.

Mycoplasmapneumoniae is the primary cause of atypical pneumonia in young adults and children, although infected patients often present with symptoms similar to a persistent influenza infection. A study performed in the U.K. (Granstrom et al, 1994) has attributed 18% of the total number of cases of acquired pneumonia in the community, to M.

SUBSTITUTE SHEET (RULE 26) 0O 97/21727 PCT/AU96/00803 2 pneumoniae infection. Periodically, M. pneumoniae is present in epidemic proportions in human communities. Previous exposure to the pathogen can result in hypersensitivity reactions upon reinfection (Cimolai et al, 1992).

Although only one in one thousand cases ofM. pneumoniae infection result in pathology of the central nervous system (CNS), infection is associated with 5-10% of cases of neurological syndromes (Koskiniemi, 1993). Typical CNS manifestations associated with infection include encephalitis, meningitis and myelitis. Additional complications associated with M. pneumoniae infection include the presence of cold agglutinins and arthropathy (Cimolai et al, 1989). A report by Zagami et al. (1994) suggests that the neurological pathology of M. pneumoniae associated encephalitis results from a cell mediated response to shared M pneumoniae antigens, or a local inflammatory response of the CNS due to the presence ofM. pneumoniae in the CNS.

However, notwithstanding the endemic and serious nature of M pneumoniae related disease, no means is available for the accurate and rapid diagnosis of M. pneumoniae infection, or for the prophylaxis of individuals exposed to infection. Accordingly, there is a clear need to develop agents useful in the diagnosis and prophylaxis of infection by mycoplasmas, in particular M. pneumoniae.

Previous attempts to develop a suitable peptide vaccine against M pneumoniae have been largely unsuccessful. For example, although hamsters immunised with M. pneumoniae may develop antibodies against the P protein of M pneumoniae, the results, in terms of the degree of humoral immunity conferred by such immunisation, have been inconsistent and unpredictable (Yayoshi et al, 1992). In particular, hamsters inoculated with the P24- SII live vaccine, which contains the P1 protein but not the 85 kDa protein, were not protected against infection. The P24-SI live vaccine, containing the P1 protein and the kDa protein, gave 50% protection against infection. The FH-P24 live vaccine, also containing both the P1 protein and the 85 kDa protein, gave 90% protection. The P24-SI and P24-SII vaccines are nitroso guanidine non-hemolysing mutants of FH-P24.

Furthermore, no success has been obtained using the 43 kDa M pneumoniae protein, SUBSTITUTE SHEET (RULE 26) 0 VVO 97/21727 PCT/AU96/00803 3 which appears to enhance the severity of infection by this pathogen (Cimolai et al, 1992).

In work leading up to the present invention, the inventors sought to develop better and more effective vaccines and diagnostic agents for M. pneumoniae, by cloning M.

pneumoniae genes encoding highly immunogenic polypeptides and homologues and derivatives thereof. The recombinant polypeptides and derivatives, homologues or analogues thereof, provide the means to develop a range of diagnostic and prophylactic agents for Mycoplasma infection which were hitherto not available.

Accordingly, one aspect of the present invention is directed to an isolated or recombinant polypeptide or a derivative, homologue or analogue thereof wherein said polypeptide is obtainable from a species of Mycoplasma.

In one embodiment of the present invention, there is provided an isolated or recombinant polypeptide or derivative, homologue or analogue thereof wherein said polypeptide is obtainable from a species of Mycoplasma and has a predicted molecular weight of approximately 16 kDa.

Preferably, the isolated or recombinant polypeptide of the present invention or a derivative, homologue or analogue thereof is further obtainable from Mycoplasma pneumoniae or M.

genitalium and M genitalium.

More preferably, said polypeptide further comprises an amino acid sequence substantially the same as the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO:4, or is at least 70% similar to all or a part thereof.

In one particularly preferred embodiment, the isolated or recombinant polypeptide or a derivative, homologue or analogue thereof is characterised by any one or more of the following properties: it is obtainable from Mycoplasmapneumoniae; SUBSTITUTE SHEET (RULE 26) O" O 97/21727 PCT/AU96/00803 4 (ii) it has a predicted molecular weight of approximately 16 kDa; or (iii) it comprises an amino acid sequence substantially as set forth in SEQ ID NO: 1 or having at least 70% similarity to all or a part thereof.

Alternatively or in addition, the isolated or recombinant polypeptide or a derivative, homologue or analogue thereof is characterised by any one of the following properties: it is obtainable from Mycoplasma genitalium; (ii) it has a predicted molecular weight of approximately 16 kDa; or (iii) it comprises an amino acid sequence substantially as set forth in SEQ ID NO:4 or having at least 70% similarity to all or a part thereof.

The homologous amino acid sequences set forth in SEQ ID Nos: 1 and 4 are only 37.3% identical overall, as described in Example 21. Accordingly, the present invention further extends to any isolated Mycoplasma polypeptide which has properties of a surface polypeptide and is at least 35% identical to SEQ ID NO: 1 or SEQ ID NO:4.

In an alternative embodiment of the present invention, there is provided an isolated polypeptide, or a derivative, homologue or analogue thereof wherein said polypeptide is obtainable from a species of Mycoplasma and wherein said polypeptide in its native form is a surface polypeptide which has adhesion properties.

Preferably, one embodiment is directed to an isolated polypeptide, or a derivative, homologue or analogue thereof wherein said polypeptide is obtainable from Mycoplasma pneumoniae, has a molecular weight of approximately 110 kDa determined by SDS/PAGE, or a predicted molecular weight of approximately 116 kDa and in its native form is a surface polypeptide which has adhesion properties.

For the present purposes, it will be understood that reference to the molecular weight of the subject polypeptide does not necessarily limit the invention, but is included especially for the purposes of nomenclature. Those skilled in the art are aware of the degree of precision associated with molecular weight estimates in respect of protein or polypeptide SUBSTITUTE SHEET (RULE 26) S 'VO 97/21727 PCT/AU96/00803 molecules and the fact that such estimates vary considerably depending upon the means employed to obtain them. For example, the subject polypeptide having derived molecular weight of 116 kDa may electrophorese on SDS/polyacrylamide gels such that it has an estimated molecular weight of only 110 kDa. Accordingly, reference herein to the term "116 kDa polypeptide" or "110 kDa polypeptide" are not to be taken as mutually exclusive definitions.

Even more particularly, this embodiment of the present invention is directed to an isolated polypeptide or derivative, homologue or analogue thereof characterised by the following properties:

(I)

(ii) (iii) (iv) (v) it is obtainable from Mycoplasmapneumoniae; it has a molecular weight of approximately 110 kDa as determined by SDS/PAGE, or a predicted molecular weight of approximately 116 kDa; it is a surface polypeptide in its native form; it has adhesion properties in its native form; or it comprises an amino acid sequence substantially as set forth in SEQ ID NO:2 or having at least 70% similarity to all or a part thereof.

Alternatively, this embodiment is directed to an isolated polypeptide, or a derivative, homologue or analogue thereof wherein said polypeptide is obtainable from Mycoplasma genitalium.

Accordingly, this alternative embodiment is directed to an isolated polypeptide or derivative, homologue or analogue thereof characterised by any of the following properties: it is obtainable from Mycoplasma genitalium; (ii) it is at least 50% identical to the amino acid sequence set forth in SEQ ID NO:2; (iii) it has the structural properties of a surface polypeptide; or (iv) it comprises an amino acid sequence substantially as set forth in SEQ ID or having at least 70% similarity to all or a part thereof.

SUBSTITUTE SHEET (RULE 26) WO 97/21727 PCT/AU96/00803 6 The present invention extends to both isolated non-recombinant polypeptides, recombinant polypeptides and isolated recombinant polypeptides ofMycoplasma described in any of the foregoing embodiments.

In particular, the present invention extends to isolated non-recombinant polypeptides, recombinant polypeptides and isolated recombinant polypeptides comprising a sequence of amino acids substantially as set forth in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:5 or having at least 70% similarity to all or a part thereof.

According to the foregoing embodiments, when the polypeptide of the present invention is a recombinant polypeptide, it may be produced in and, if desirable isolated from, any virus particle or cell. As will be known to those skilled in the relevant art, a cell for production of a recombinant polypeptide is selected on the basis of several parameters including the genetic constructs used to express the polypeptide under consideration, stability and activity of said polypeptide. It will also be known to those skilled in the art, that the stability or activity of a recombinant polypeptide may be determined, at least in part, by post-translational modifications to the polypeptide, for example glycosylation, acylation or alkylation reactions, amongst others, which may vary between cell lines used to produce the recombinant polypeptide.

The present invention extends further to a recombinant polypeptide according to any of the foregoing embodiments or a derivative, homologue or analogue thereof, wherein said polypeptide is produced in any virus particle or a prokaryotic or eukaryotic cell or a culture thereof.

In a preferred embodiment, the present invention extends to a recombinant polypeptide according to any of the foregoing embodiments or a derivative, homologue or analogue thereof, wherein said polypeptide is produced in a bacterial cell or culture thereof belonging to the genus Mycoplasma, in particular a cell of M. pneumoniae or M.

genitalium or a culture thereof or an Escherichia coli cell.

SUBSTITUTE SHEET (RULE 26) 'WO 97/21727 PCT/AU96/00803 7 The term "polypeptide" as used herein shall be taken to refer to any polymer consisting of amino acids linked by covalent bonds and includes within its scope full-length proteins and parts or fragments thereof, for example oligopeptides and short peptide sequences consisting of at least two amino acid residues. Also included within the scope of the definition of a "polypeptide" are amino acid sequence variants, containing amino acid substitutions, deletions, or insertions which do not alter the essential properties of said polypeptide, for example its immunogenicity or effectiveness as a peptide vaccine against Mycoplasma ssp, amongst others. Accordingly, a polypeptide may be isolated from a source in nature, or chemically synthesized. Furthermore, a polypeptide may be derived from a full-length protein by chemical or enzymatic cleavage, using reagents such as CNBr, trypsin, or chymotrypsin, amongst others.

The term "recombinant polypeptide" as used herein shall be taken to refer to a polypeptide which is produced in a virus particle or a cell by the expression therein of a genetic sequence encoding said polypeptide under the control of a suitable promoter, wherein a genetic manipulation has been performed in order to achieve said expression. Genetic manipulations will be known to those skilled in the art and include, but are not limited to nucleic acid isolation, digestion, ligation, amplification, hybridisation or sequencing.

The term "surface polypeptide" or similar term as used herein shall be taken in its broadest context to refer to a polypeptide which is localised on, or intrinsically or extrinsically associated with, the surface layer ofMycoplasma spp. and in particular M pneumoniae or M. genitalium. A surface polypeptide, or at least an epitope thereof, is accessible to recognition by the immune system of the host organism without lysis of the infecting pathogen.

The term "adhesion properties" as used herein shall be taken to refer to a functional characteristic of a polypeptide which facilitates the association, adherence or attachment of a micro-organism to a cell of a host organism during the infectious phase. In the present context, adhesion properties of the micro-organism usually render M. pneumoniae capable of binding host cells of the upper respiratory tract.

SUBSTITUTE SHEET (RULE 26) WO 97/21727 PCT/AU96/00803 8 The term "native form" or "native state" or similar term as used herein with reference to a characteristic of a polypeptide, shall be taken as a reference to the inherent properties of a non-recombinant polypeptide when it is present in the cell from which it originates, such as a mycoplasma cell, in particular aM pneumoniae cell. For example, the M pneumoniae polypeptide set forth in SEQ ID'NO:2 has a predicted molecular weight of 116 kDa and is said to be in its native state when it is in a M. pneumoniae cell and no genetic manipulations have been performed upon it. Accordingly, said polypeptide when present in a M pneumoniae cell, has the inherent properties of being localised to the cell surface and is an adhesion polypeptide.

The term "predicted molecular weight" as used herein in relation to a polypeptide refers to a molecular weight which is determined by a summation of the molecular weights of individual chemical elements or atoms comprised therein.

In a more particularly preferred embodiment, the present invention extends to a recombinant polypeptide or a derivative, homologue or analogue thereof produced in a virus particle, prokaryotic or eukaryotic cell or a virus or cell culture thereof, wherein said recombinant polypeptide has an amino acid sequence which is substantially the same as the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2 or a part thereof.

It is understood in the art that although parts or fragments of a full-length protein may not possess all of the properties of the full-length protein, they are at least useful as diagnostic reagents, or as immunogens in the production of immunoreactive molecules or vaccine preparations. Accordingly, the present invention extends to parts or fragments of an isolated polypeptide defined according to the embodiments herein.

In a particularly preferred embodiment, the present invention extends to a part or fragment of the isolated or recombinant form of a polypeptide set forth in SEQ ID NO: 1 or SEQ ID NO:2.

In a more particularly preferred embodiment of the invention, there is provided a fragment SUBSTITUTE SHEET (RULE 26) WO~ 97/21727 PCT/AU96/00803 9 or derivative of the amino acid sequence of the forth in SEQ ID NO: 2 comprising amino acid residues 9 to 473, 467 to 709, 709 to 850, 846 to 896, 887 to 962 or 969 to 1029 of said amino acid sequence or a homologue, analogue or derivative thereof. The specific fragments or derivatives of SEQ ID NO: 2 to which this embodiment of the invention relates are exemplified in Table 5 which is incorporated herein.

Even more particularly preferred, the fragment or derivative of SEQ ID NO: 2 is highly immunogenic or antigenic, preferably when compared to the full-length amino acid sequence. Those skilled in the art will be aware that such peptide derivatives or fragments are useful as immunogens which comprise a B cell or T cell epitope. Particularly suitable for this purpose is a fragment or derivative of SEQ ID NO: 2 comprising amino acid residues 9 to 473 of SEQ ID NO: 2 or a homologue, analogue or derivative thereof.

The polypeptide, or a derivative, homologue or analogue thereof of the present invention is particularly useful as a polypeptide component in a vaccine composition which is designed to protect an individual against infection by a species of Mycoplasma, in particular to protect an individual against infection by Mycoplasma pneumoniae or M.

genitalium. The essential feature of said polypeptide, or derivative, homologue or analogue thereof for the present purpose is whether it is "immunogenic", defined hereinafter as the ability of said polypeptide, or a derivative, homologue or analogue thereof, to elicit B cell and/or T cell responses in the host, as part of the immunization process. It is understood by a person skilled in the art that not all parts of a polypeptide or derivative, homologue or analogue thereof are equally immunogenic. In fact, a polypeptide or a derivative, homologue or analogue thereof may be comprised of several different overlapping, or non-overlapping regions, which, in isolation, or in combination, are highly immunogenic. Said highly immunogenic region, or a derivative, homologue or analogue thereof, is hereinafter referred to as a "B cell and/or T cell epitope".

Furthermore, a B cell or T cell epitope of a polypeptide or a derivative, homologue or analogue thereof may comprise any one or more of the following: the primary amino acid sequence of said region, known in the art as a SUBSTITUTE SHEET (RULE 26) WO 97/21727 PCT/AU96/00803 continuous non-conformational epitope; (ii) the secondary structure which said region adopts, known in the art as a continuous conformational epitope; (iii) the tertiary structure which said region adopts in contact with another region of the same polypeptide molecule, known in the art as a discontinuous conformational epitope; or (iv) the quaternary structure which said region adopts in contact with a region of another polypeptide molecule, known in the art as a discontinuous conformational epitope.

Accordingly, immunogenic polypeptides or derivatives, homologues or analogues thereof comprising the same, or substantially the same primary amino acid sequence are hereinafter defined as "immunogens which comprise a B cell or T cell epitope", or similar term.

Immunogenic polypeptides or derivatives, homologues, or analogues thereof comprising different primary amino acid sequences may comprise immunologically identical immunogens, because they possess conformational B cell or T cell epitopes that are recognised by the immune system of a host species to be identical. Such immunogenic polypeptides or derivatives, homologues or analogues thereof are hereinafter defined as "immunogens which mimic or cross-react with a B cell or T cell epitope", or similar term.

Accordingly, the present invention extends to an immunogen which comprises, mimics, or cross-reacts with a B-cell or T-cell epitope of an isolated or recombinant polypeptide according to any of the foregoing embodiments or a derivative, homologue or analogue thereof. In a particularly preferred embodiment, the present invention provides an immunogen which comprises, mimics, or cross-reacts with a B-cell or T-cell epitope of an isolated or recombinant polypeptide which in its native form is obtainable from a species ofMycoplasma such as, but not limited to M pneumoniae and has a predicted molecular weight of approximately 16 kDa or approximately 116 kDa or a homologue, analogue or derivative thereof. According to this embodiment, when the polypeptide has a predicted SUBSTITUTE SHEET (RULE 26) *0 97/21727 PCT/AU96/00803 11 molecular weight of approximately 116 kDa it is also preferred that said polypeptide in its native form is a surface polypeptide with adhesion properties.

In a particularly preferred embodiment of the invention, there is provided an immunogen comprising amino acid residues 9 to 473, 467 to 709, 709 to 850, 846 to 896, 887 to 962 or 969 to 1029 of SEQ ID NO: 2 or a homologue, analogue or derivative thereof. More particularly, the preferred immunogen, according to this embodiment of the invention, comprises amino acid residues 9 to 473 of SEQ ID NO: 2 or a homologue, analogue or derivative thereof.

In an alternative embodiment, the present invention extends to an immunogen which comprises, mimics, or cross-reacts with a B-cell or T-cell epitope of a polypeptide according to any of the foregoing embodiments or a derivative, homologue or analogue thereof, wherein said polypeptide comprises a sequence of amino acids which is at least about 40% similar to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2, more preferably at least about 60% similar, still more preferably at least about 80% similar and even still more preferably, at least about 99% similar to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.

In the present context, "homologues" of a polypeptide refer to those polypeptides, enzymes or proteins which have similar properties as a polypeptide of the present invention, for example surface protein adhesion properties or immunogenic properties as described supra, notwithstanding any amino acid substitutions, additions or deletions thereto.

Furthermore, amino acids may be replaced by other amino acids having similar properties, for example hydrophobicity, hydrophilicity, hydrophobic moment, antigenicity, propensity to form or break a-helical structures or P-sheet structures, and so on.

The present invention clearly extends to such amino acid variants, provided that such molecules still function as B cell or T-cell epitopes capable of mediating an immune SUBSTITUTE SHEET (RULE 26) WO~ 97/21727 PCT/AU96/00803 12 response or functioning as a surface polypeptide. Preferably, a homologue will still function as a peptide immunogen which mimics or cross-reacts to B-cell or T-cell epitopes of a Mycoplasma spp. polypeptide of the present invention.

Furthermore, a homologue may be isolated or derived from the same or another Mycoplasma species. Preferred sources of homologues of a Mycoplasma pneumoniae polypeptide according to the present invention are M. genitalium, M. penetrans, M.

iowae, M. gallisepticum, M. imitans, M. muris, M. urealyticum or M. pirum, amongst others. In a particularly preferred embodiment of the invention, homologues of the M.

pneumoniae 16 kDa and 116 kDa polypeptide are isolated from M. genitalium. The amino acid sequence of the M. genitalium homologue of the 16 kDa polypeptide is set forth in SEQ ID NO:4. The amino acid sequence of the M. genitalium homologue of the 116 kDa polypeptide is set forth in SEQ ID Substitutions encompass amino acid alterations in which an amino acid is replaced with a different naturally-occurring or a non-conventional amino acid residue. Such substitutions may be classified as "conservative", in which case an amino acid residue contained in a repressor polypeptide is replaced with another naturally-occurring amino acid of similar character, for example Gly++Ala, Val++IlehLeu, Asp++Glu, Lys-+Arg, Asn-+Gln or Phet+Trp-+Tyr.

Substitutions encompassed by the present invention may also be "non-conservative", in which an amino acid residue is substituted with an amino acid having different properties, such as a naturally-occurring amino acid from a different group (eg. substituted a charged or hydrophobic amino acid with alanine), or alternatively, in which a naturally-occurring amino acid is substituted with a non-conventional amino acid.

Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide; insertions will usually be of the order of about 1-10 amino acid residues; and deletions will range from about 1-20 residues.

SUBSTITUTE SHEET (RULE 26) 0o 97/21727 PCT/AU96/00803 13 Amino acid alterations to the peptides contemplated herein include insertions such as amino acid and/or carboxyl terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than amino or carboxyl terminal fusions, of the order of about 1 to 4 residues.

Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein. Deletional variants are characterised by the removal of one or more amino acids from the sequence. Substitutional variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. Such substitutions may be made in accordance with Table 1.

The amino acid variants referred to in Table 1 may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulations. Techniques for making substitution mutations at predetermined sites in DNA having known sequence are well known, for example through M13 mutagenesis. The manipulation of DNA sequences to produce variant proteins which manifest as substitutional, insertional or deletional variants are well known in the art.

SUBSTITUTE SHEET (RULE 26) VO 97/21727 PCT/AU96/00803 14 TABLE 1 Amino Acid Three-letter One-letter Abbreviation Symbol Alanine Ala

A

Arginine Arg

R

Asparagine Asn

N

Aspartic acid Asp

D

Cysteine Cys

C

Glutamine Gin

Q

Glutamic acid Glu

E

Glycine Gly

G

Histidine His

H

Isoleucine Ile

I

Leucine Leu

L

Lysine Lys

K

Methionine Met

M

Phenylalanine Phe

F

Proline Pro

P

Serine Ser

S

Threonine Thr

T

Tryptophan Trp

W

Tyrosine Tyr

Y

Valine Val

V

"Analogues" encompass polypeptides which are functionally equivalent or at least have similar properties as a polypeptide of the present invention, notwithstanding the occurrence of any non-naturally occurring or modified amino acid residues therein. Nonnaturally occurring amino acid residues contemplated in an analogue of the present invention are set forth in Table 2.

SUBSTITUTE SHEET (RULE 26) WO 97/21727 WO 9721727PCT/AU96/00803 TABLE 2 Non-conventional Code Non-conventional Code amino acid amino acid ix-aminobutyric acid rz-amino-ix-methylbutyrate aminocyclopropanecarboxylate aminoisobutyric acid aminonorbornylcarboxylate cyclohexylalanine cyclopentylalanine D-alanine D-arginine D-aspartic acid D-cysteine D-glutamine D-glutamic acid D-histidine D-isoleucine D-leucine D-Iysine D-methiomine D-ornithine D-phenylalanine D-proline D-serine D-threonine D-tryptophan D-tyrosine Abu Mgabu Cpro Aib Norb Chexa Cpen Dal Darg Dasp Dcys Dgln Dglu Dhis Dile Dleu Dlys Dmet Dorn Dphe Dpro Dser Dthr Dtrp Dtyr L-N-methylalanine L-N-methylarginine L-N-methylasparagine L-N-methylaspartic acid L-N-methylcysteine L-N-methylglutamine L-N-methylglutamic acid L-N-methylhistidine L-N-methyl isolleucine L-N-methylleucine L-N-methyllysine L-N-methylmethionine L-N-methylnorleucine L-N-methylnorvaline L-N-methylornithine L-N-methylphenylalanine L-N-methylproline L-N-methylseriie L-N-methylthreonine L-N-methyltryptophan L-N-methyltyrosine L-N-methylvaline L-N-methylethylglycine L-N-methyl-t-butylglycine L-norleucine L-norvaline a-methyl-aminoisobutyrate Nmala Nmarg Nmasn Nmasp Nmcys Nmgln Nmglu Nmhis Nmile Nmleu Nmlys Nmmet Nmnle Nmnva Nmorn Nmphe Nmpro Nmser Nmthr Nmtrp Nmtyr Nmval Nmetg Nmtbug NMe Nva Maib SUBSTITUTE SHEET (RULE ~WO 97/2 1727 PTA9/00 PCT/AU96/00803 D-valine D-rc-methylalanine D-a-methylarginine D-rz-methylasparagine D-a-methylaspartate D-a-methylcysteine D-a-methylglutaniine D-a-methylhistidine D-a-methylisoleucine D-a-methylleucine D-cz-methyllysine D-a-methylmethionine D-a-methylornithine D-a-methylphenylalanine D-a~-meffiylproline D-a-methylserine D-a-methyhthreonine D-a-methyltryptophan D-a-methyltyrosine D-a-methylvaline D-N-methylalanine D-N-methylarginine D-N-methylasparagine D-N-methylaspartate D-N-methylcysteine D-N-methylglutamine D-N-methylglutatnate D-N-methylhistidine D-N-methylisoleucine D-N-methylleucine D-N-methyllysine Dval Dmala Dmarg Dmasn Dmasp Dmcys Dmgln Dmhis Dmile Dmleu Dmlys Dmmet Dmorn Dmphe Dmpro Dmser Dmthr Dmtrp Dmty Dmval Dnmala Dnmarg Dnmasn Dnmasp Dnmcys Dnmgln Dnmglu Dnmbis Dnmile Dnmleu Dnmlys a-methyl-y-aninobutyrate a-methyleyelohexylalanine a-methylcylcopentylalanine ca-methyl-ct-napthylalanine c-methylpenicillamine N-(4-aminobutyl)glycine N-(2-aminoethyl)glycine N-(3-aminopropyl)glycine N-amino-a-methylbutyrate a-napthylalanine N-benzylglycine N-(2-carbamylethyl)glycine N-(carbamylmethyl)glycine N-(2-carboxyethyl)glycine N-(carboxymethyl)glycine N-cyclobutylglycine N-cycloheptylglycine N-cyclohexylglycine N-cyclodecylglycine N-cylcododecylglycine N-cyclooctylglycine N-cyclopropylglycine N-cycloundecylglycine N-(2,2-diphenylethyl) glycine N-(3 ,3-diphenylpropyl) glycine N-(3-guanidinopropyl) glycine 1-hydroxyethyl)glycine N-(hydroxyethyl))glycine N-(imidazolylethyl)) glycine N-(3-indolylyethyl) glycine N-methyl-y-aminobutyrate Mgabu Mchexa Mcpen Manap Mpen Nglu Naeg Norn Nmaabu Anap Nphe Ngin Nasn Nglu Nasp Ncbut Nchep Nchex Ncdec Ncdod Ncoct Ncpro Ncund Nbhm Nbhe Narg Nthr Nser Nhis Nhtrp Nmgabu SUBSTITUTE SHEET (RULE 26) 'WO97/21727. PTA9/00 PCT/AU96/00803 N-methylcyclohexylalanine D-N-methylornithine N-methylglycine N-methylaminoisobutyrate N-(l1-methylpropyl)glycine N-(2-methylpropyl)glycine D-N-methyhryptophan D-N-methyltyrosine D-N-methylvaline y-aminobutyric acid L-t-butylglycine L-ethylglycine L-homophenylalanine L-ea-methylarginine L-ca-methylaspartate L-z-methylcysteine L-a-methylglutamine L-a-methylhistidine L-iz-methylisoleucine L-a-methylleucine L-ea-methylmethionine L-a-methylnorvaline L-a-methylphenylalanine L-a-methylserine L-n-methyltryptophan L-a-methylvaline ,2-diphenylethyl) carbamylmethyl)glycine 1 -carboxy-1I -(2,2-diphenylethylamino)cyclopropane Nmchexa D-N-methylmethionine Dnmorn N-methylcyclopentylalanine Nala D-N-methylphenylalanine Nmaib D-N-methylproline Nile D-N-methylserine Nleu D-N-methylthreonine Dnmtrp N-(l1-methylethyl)glycine Dnmtyr N-methyla-napthylalanjne Dnmval N-methylpenicillamine, Gabu N-(p-hydroxyphenyl)glycine Thug N-(thiomethyl)glycine Etg penicillamnine Hphe L-a-methylalanine Marg L-a-methylasparagine Masp L-a-methyl-t-butylglycine Mcys L-methylethylglycine Mgln L-a-methylglutamate Mhis L-a-methylhomo phenylalanine Mile N-(2-methylthioethyl) glycine, Mleu L-cz-methyllysine Mmet L-a-methylnorleucine Mnva L-a-methylornithine Mphe L-rc-methylproline Mser L-a-methylthreonine Mtrp L-a-methyltyrosmne Mval L-N-methylhomo phenylalanine ,3-diphenylpropyl) Nnbhm carbamylmethyl)glycine Nmnbc Dnrnmet Nmcpen Dnmphe Dnmpro Dnmser Dnmthr Nval Nmanap Nmpen Nhtyr Ncys Pen Mala Masn Mtbug Metg Mglu Mhphe Nmet Mlys Mnle Morn Mpro Mthr Mtyr Nmhphe Nnbhe SUBSTITUTE SHEET (RULE 26) WO 97/21727 PCT/AU96/00803 18 The term "derivative" in relation to a polypeptide as hereinbefore defined shall be taken to refer hereinafter to mutants, parts or fragments of a functional molecule. Derivatives also include modified peptides in which ligands are attached to one or more of the amino acid residues contained therein, such as carbohydrates, enzymes, proteins, polypeptides or reporter molecules such as radionuclides or fluorescent compounds. Glycosylated, fluorescent, acylated or alkylated forms of the subject polypeptides are particularly contemplated by the present invention. Additionally, derivatives of a polypeptide as hereinbefore defined may comprise fragments or parts of an amino acid sequence disclosed herein and are within the scope of the invention, as are homopolymers or heteropolymers comprising two or more copies of the subject polypeptides. Procedures for derivatizing peptides are well-known in the art.

Particularly preferred derivatives of a polypeptide according to the present invention include amino acid residues 9 to 473, 467 to 709, 709 to 850, 846 to 896, 887 to 962 or 969 to 1029 of the amino acid sequence set forth in SEQ ID NO:2. These derivative polypeptides are exemplified in Table 5 herein.

In a most particularly preferred embodiment of the invention, the derivative is useful as an immunogen to elicit the production of antibodies capable of recognising Mycoplasma pneumoniae or at least a protein component thereof. According to this embodiment, there is provided a recombinant polypeptide derivative of SEQ ID NO:2 comprising amino acids 9 to 473 thereof.

The present invention extends further to derivatives, homologues or analogues of the derivative polypeptides provided herein, which are at least useful as immunogens.

Other examples of recombinant or synthetic mutants and derivatives of the peptide immunogens of the present invention include single or multiple substitutions, deletions and/or additions to any molecule associated with the ligand such as carbohydrates, lipids SUBSTITUTE SHEET (RULE 26) W iO 97/21727 PCT/AU96/00803 19 and/or proteins or polypeptides. Naturally occurring or altered glycosylated or acylated forms of the subject peptides are particularly contemplated by the present invention.

Additionally, homopolymers or heteropolymers comprising one or more copies of the subject peptide listed in SEQ ID NO: 1 or SEQ ID NO:2, or one or more derivatives, homologues or analogues thereof, are within the scope of the invention.

The immunogen of the present invention as described supra or a derivative, homologue or analogue thereof is useful in vaccine compositions and/or as an antigen to elicit polyclonal and monoclonal antibody production and/or in the detection of antibodies against M.

pneumoniae in infected individuals.

To improve the immunogenicity of a subject polypeptide of the present invention one or more amino acids not corresponding to the original protein sequence may be added to the amino or carboxyl terminus of the polypeptide. Such extra amino acids are useful for coupling the polypeptides to another peptide or polypeptide, to a large carrier protein or to a solid support. Amino acids that are useful for these purposes include but are not limited to tyrosine, lysine, glutamic acid, aspartic acid, cysteine and derivatives thereof.

Additional protein modification techniques may be used, NH 2 -acetylation or COOHterminal amidation, to provide additional means for coupling the polypeptides to another polypeptide, protein, or peptide molecule, or a support. Procedures for coupling polypeptides to each other, carrier proteins and solid supports are well known in the art.

Furthermore, the polypeptide may be immobilised to a polymeric carrier or support material which possesses immunogenic properties. Polypeptides containing the abovementioned extra amino acid residues at either the carboxyl- or amino- termini and either uncoupled or coupled to a carrier or solid support, are consequently within the scope of the present invention.

In an alternative embodiment, the immunogenicity of a polypeptide immunogen may be improved using molecular biology techniques to produce a fusion protein containing one or more of the polypeptide of the present invention and a highly immunogenic protein. For example, fusion proteins containing a polypeptide which is of low immunogenicity and the SUBSTITUTE SHEET (RULE 26) VWO 97/21727 PCT/AU96/00803 highly immunogenic B subunit of cholera toxin may induce an immune response to the polypeptide. The present invention also contemplates the use of genes encoding cytokines, for example interleukin, in fusion with the subject polypeptide immunogen.

Preferably, the polypeptide immunogen or a derivative, homologue or analogue thereof when administered to a mammal mediates an immune response in said mammal. More preferably, the immunogen of the present invention when administered to a mammal, induces humoral immunity against Mycoplasma spp. in particular M. pneumoniae or M.

genitalium in said primate. Still more preferably, the immunogen when administered, prevents the onset, development or progression, of symptoms associated with Mycoplasma pneumoniae infections, for example atypical pneumonia, or lung lesions, or inflammation of the respiratory tract, or inflammation of the central nervous system, amongst others.

The invention further encompasses functionally equivalent variants, derivatives, homologues or analogues of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2, which do not significantly reduce the immunogenic and/or antigenic properties of said polypeptide. Such functionally equivalent derivatives and homologues are as described supra. The invention also encompasses homopolymers or heteropolymers of one or more of the polypeptides set forth in SEQ ID NO: 1 or SEQ ID NO:2, and derivatives, homologues or analogues thereof are within the scope of the invention. Also within the scope of this invention are polypeptides of fewer amino acid residues than the subject polypeptides but which encompass one or more immunogenic epitopes present in any one of the polypeptides and thus retain the immunogenic and/or antigenic properties of the base polypeptide.

The use of polypeptide analogues can result in polypeptides with increased immunogenic and/or antigenic activity, that are less sensitive to enzymatic degradation, and which are more selective. A suitable proline analogue is 2-aminocyclopentane carboxylic acid (PAcSc) which has been shown to increase the immunogenic activity of a native polypeptide more than 20 times (Mierke et al., 1990; Portoghese et al., 1990; Goodman et al., 1987).

SUBSTITUTE SHEET (RULE 26) k0 97/21727 PCT/AU96/00803 21 In a related embodiment, the present invention provides a substantially homogeneous form of any one or more polypeptide immunogens selected from the list comprising SEQ ID NO: 1 and SEQ ID NO:2 or a derivative, homologue or analogue thereof, wherein the term "substantially homogeneous" is defined herein as being in a form suitable for interaction with an immunologically interactive molecule. Preferably, the immunogen is at least homogeneous, more preferably at least 75% homogeneous and yet still more preferably at least about 95-100% homogeneous, in terms of percentage purity on a weight-for-weight basis.

Accordingly, the present invention extends to a method of purifying an polypeptide immunogen of the present invention, said method comprising a combination of Triton X- 114 partitioning and size separation techniques, amongst others. In particular, the M.

pneumoniae polypeptide set forth in SEQ ID NO:2 is purified by Triton X-114 partitioning and SDS/polyacrylamide gel electrophoresis as described herein, in Examples 1 to 4 inclusive. Methods of purification of said polypeptide utilising additional or alternative procedures, for example reverse phase chromatography, ion-exchange chromatography, or affinity chromatography are also contemplated.

The present invention contemplates further a method of isolation of the polypeptide set forth in SEQ ID NO: 1, said method comprising any combination of purification procedures selected from the list comprising chromatographic, phase separation, electrophoresis, ionexchange chromatography, gel filtration, reverse-phase chromatography, SDS/polyacrylamide gel electrophoresis or detergent partitioning, amongst others. It will be known to those skilled in the art how to vary the above procedures.

A further aspect of the present invention provides a vaccine composition comprising a polypeptide component which comprises an isolated immunogenic polypeptide obtainable or derived from a species of Mycoplasma, or alternatively, a recombinant immunogenic polypeptide comprising an amino acid sequence similar or identical to said isolated immunogenic polypeptide, in combination with a pharmaceutically acceptable carrier or diluent.

SUBSTITUTE SHEET (RULE 26) WO 97/21727 PCT/AU96/00803 22 Preferably, the polypeptide component of said vaccine composition is a polypeptide according to any of the foregoing embodiments described herein, in particular the M pneumoniae orM genitalium polypeptides set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO: 4 or SEQ ID NO: 5 or a derivative, homologue or analogue thereof.

The vaccine composition of the present invention is effective in mediating an immune response when ingested, injected, or otherwise administered to a mammal. In a preferred embodiment, said vaccine induces humoral immunity against a Mycoplasma spp., in particular M pneumoniae orM genitalium, when injected, or otherwise administered to a mammal. More preferably, said vaccine composition prevents the onset, development, or progression of symptoms associated with M pneumoniae infection, for example atypical pneumonia, lung lesions, inflammatory reactions of the respiratory tract or central nervous system, amongst others.

The vaccine composition of present invention extends to vaccines in which the polypeptide component comprises a variant of the polypeptides referred to supra. The term "variant" as used herein shall be taken to include a mutant, derivative, part, fragment, analogue, or homologue of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2 or SEQ ID NO: 4 or SEQ ID NO: 5, which is at least about 30% similar, more preferably at least about 70% similar, still more preferably at least about 80% similar and even still more preferably at least about 99% similar to all, or a part, of the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO: 4 or SEQ ID NO: In an alternative embodiment the present invention provides a vaccine composition comprising an isolated or recombinant immunogenic polypeptide which is obtainable from a species of Mycoplasma, in combination with a pharmaceutically acceptable carrier or diluent, wherein said polypeptide is further characterised by any of the following properties or is derived from a polypeptide having any of the following properties: it has a predicted molecular weight of approximately 16 kDa; (ii) it has a molecular weight of approximately 110 kDa as determined by SDS/PAGE, or a predicted molecular weight of approximately 116 kDa; SUBSTITUTE SHEET (RULE 26) VVO 97/21727 PCT/AU96/00803 23 (iii) it is a surface polypeptide in its native form; (iv) it has adhesion properties in its native form; or it comprises an amino acid sequence substantially as set forth in SEQ ID NO: 1 or SEQ ID NO:2 or SEQ ID NO: 4 or SEQ ID NO: 5 or having at least 70% similarity to all or a part thereof.

In a particularly preferred embodiment the present invention provides a recombinant vaccine, which vaccine comprises: a recombinant polypeptide comprising a sequence of amino acids as set forth in either SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO: 4 or SEQ ID NO: 5 or a derivative, homologue or analogue thereof which is capable of mediating an immune response against M. pneumoniae or M. genitalium; and (ii) a pharmaceutically acceptable carrier or diluent; According to this aspect of the invention, said vaccine mediates an immune response against Mycoplasma spp., in particular M pneumoniae or M genitalium, when the vaccine is injected, or otherwise administered to a mammal, for example a primate such as a human or monkey or a rodent such as a mouse, rat, hamster or guinea pig. Still more preferably, the vaccine induces humoral immunity against M pneumoniae or M. genitalium in said mammal. Even still more preferably, the recombinant vaccine of the present invention prevents the onset, development, or progression of symptoms associated with M.

pneumoniae infection, for example atypical pneumonia, lung lesions, inflammatory reactions of the respiratory tract, or of the central nervous system, amongst others.

In a further preferred embodiment, the vaccine may also comprise an adjuvant to boost the immune response of an animal to the immunogenic polypeptide when the vaccine is administered to said animal.

A third aspect of the present invention provides a method of producing a vaccine composition which method comprises the steps of: SUBSTITUTE SHEET (RULE 26) \VO 97/21727 PCT/AU96/00803 24 diluting a substantially homogeneous form of a polypeptide immunogen comprising a M. pneumoniae or M. genitalium polypeptide according to any of the embodiments described herein, in particular the M. pneumoniae polypeptide set forth in SEQ ID NO: 1 or SEQ ID NO:2 or a derivative, homologue or analogue thereof or the M. genitalium polypeptide set forth in SEQ ID NO: 4 or SEQ ID NO: 5, in a pharmaceutically acceptable carrier or diluent; and (ii) optionally, combining said polypeptide immunogen or a derivative, homologue or analogue thereof with a physiologically acceptable adjuvant.

Preferably, the method according to this aspect of the invention comprises the further first step of preparing a substantially homogeneous form of a polypeptide immunogen comprising a M pneumoniae polypeptide according to any of the embodiments described herein, in particular theM pneumoniae polypeptide set forth in SEQ ID NO: 1 or SEQ ID NO:2 or the M. genitalium polypeptide set forth in SEQ ID NO: 4 or SEQ ID NO: 5 or a derivative, homologue or analogue thereof, which is capable of mediating an immune response against M pneumoniae or M genitalium.

More preferably, said method of producing a vaccine composition comprises the further first step of culturing a micro-organism, bacterial cell, virus particle, fungal cell, insect cell, yeast cell, plant cell, or animal cell which comprises a nucleic acid molecule contained therein which encodes, or is complementary to a nucleic acid molecule which encodes a recombinantM. pneumoniae polypeptide immunogen according to any of the embodiments described herein for a time and under conditions sufficient for expression of said nucleic acid molecule to occur to produce said immunogen.

Even more preferably, said method comprises the further first step of transfecting, transforming or otherwise introducing said nucleic acid molecule into a micro-organism, bacterial cell, virus particle, fungal cell, yeast cell, insect cell, plant cell, or animal cell.

The term "mediating an immune response" as hereinbefore described is defined in its SUBSTITUTE SHEET (RULE 26) WvO 97/21727 PCT/AU96/00803 broadest context to include the elicitation of T-cell activation by a polypeptide, and/or the generation, by B-cells of antibodies which cross-react with one or more polypeptide immunogen molecules of the present invention.

According to these embodiments of the present invention, said polypeptide immunogen includes a polypeptide which comprises, mimics, or cross-reacts with a B cell or T cell epitope of any polypeptide according to the embodiments described herein or a derivative, homologue or analogue thereof.

Preferably, said polypeptide immunogen includes any polypeptide comprising a sequence of at least 10 amino acid residues in length, which are substantially the same as any part of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2 or SEQ ID NO: 4 or SEQ ID NO: 5, or a derivative, analogue, or homologue thereof, as described supra.

Particularly preferred derivatives according to this embodiment are the derivatives of the 116 kDa M pneumoniae polypeptide (SEQ ID NO: 2) which are listed in Table 5. In a most particularly preferred embodiment, the derivative comprises amino acid residues 9 to 473 of SEQ ID NO: 2 or a homologue, analogue or derivative thereof.

A further aspect of the present invention provides an immunologically interactive molecule prepared against a polypeptide immunogen which comprises, cross-reacts or mimics a Bcell or T-cell epitope of an M pneumoniae polypeptide, in particular a polypeptide comprising a sequence of amino acids set forth in SEQ ID NO:1 or SEQ ID NO:2 or a derivative, homologue or analogue thereof according to any of the embodiments described herein.

The term "immunologically interactive molecule" is herein defined as polyclonal or monoclonal antibodies, or functional derivatives thereof, for example Fabs, SCABS (single-chain antibodies) or antibodies conjugated to an enzyme, radioactive or fluorescent tag, the only requirement being that said immunologically interactive molecule is capable of binding to a polypeptide obtainable from Mycoplasma spp., or a derivative, homologue SUBSTITUTE SHEET (RULE 26) S O 97/21727 PCT/AU96/00803 26 or analogue thereof, or to a molecule which mimics the 3-dimensional structure of same.

In the present context, it is preferred that an immunologically interactive molecule is capable of binding to a polypeptide which comprises a sequence of amino acids set forth in SEQ ID NO: 1 or SEQ ID NO:2 or SEQ ID NO: 4 or SEQ ID NO: 5 or a derivative, homologue or analogue thereof, or a molecule which mimics the 3-dimensional structure of same.

Preferably, the immunologically interactive molecule of the present invention is prepared against an isolated or recombinant polypeptide of M. pneumoniae or M. genitalium according to any embodiment described herein.

Conventional methods can be used to prepare the immunologically interactive molecules.

For example, by using a polypeptide of the present invention polyclonal antisera or monoclonal antibodies can be made using standard methods. As demonstrated in Examples 1 and 2 of the present invention, a mammal, a mouse, hamster, or rabbit) can be immunized with an immunogenic form of the polypeptide which elicits an antibody response in the mammal. Techniques for conferring immunogenicity on a polypeptide include conjugation to carriers or other techniques well known in the art. For example, the polypeptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titres in plasma or serum.

Standard ELISA or other immunoassay can be used with the immunogen as antigen to assess the levels of antibodies. Following immunization, antisera can be obtained and, if desired IgG molecules corresponding to the polyclonal antibodies may be isolated from the sera.

To produce monoclonal antibodies, antibody producing cells (lymphocytes) can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells. Such techniques are well known in the art. For example, the hybridoma technique originally developed by Kohler and Milstein (1975) as well as other techniques such as the human B-cell hybridoma technique (Kozbor et al., 1983), the EBV-hybridoma technique to SUBSTITUTE SHEET (RULE 26) 'WO 97/21727 PCT/AU96/00803 27 produce human monoclonal antibodies (Cole et al., 1985), and screening of combinatorial antibody libraries (Huse et al., 1989). Hybridoma cells can be screened immunochemically for production of antibodies which are specifically reactive with the polypeptide and monoclonal antibodies isolated.

As with all immunogenic compositions for eliciting antibodies, the immunogenically effective amounts of the polypeptides of the invention must be determined empirically.

Factors to be considered include the immunogenicity of the native polypeptide, whether or not the polypeptide will be complexed with or covalently attached to an adjuvant or carrier protein or other carrier and route of administration for the composition, i.e.

intravenous, intramuscular, subcutaneous, etc., and the number of immunizing doses to be administered. Such factors are known in the vaccine art and it is well within the skill of immunologists to make such determinations without undue experimentation.

The term "antibody" as used herein, is intended to include fragments thereof which are also specifically reactive with a polypeptide which comprises, mimics, or cross-reacts with a B cell or T cell epitope of a Mycoplasma polypeptide according to the embodiments described herein, in particular a polypeptide set forth in SEQ ID NO: 1 or SEQ ID NO:2 or SEQ ID NO: 4 or SEQ ID NO: 5 or a homologue, analogue or derivative thereof.

Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab')2 fragments can be generated by treating antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments.

It is within the scope of this invention to include any second antibodies (monoclonal, polyclonal or fragments of antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available anti-immunoglobulin antibody.

The polyclonal, monoclonal or chimeric monoclonal antibodies can be used to detect the polypeptide of the invention or a derivative, homologue or analogue thereof in various SUBSTITUTE SHEET (RULE 26) VO 97/21727 PCT/AU96/00803 28 biological materials, for example they can be used in an ELISA, radioimmunoassay or histochemical tests. Thus, the antibodies can be used to test for binding to a polypeptide of the invention, or a derivative, homologue or analogue thereof in a sample and in order to determine B cell or T cell epitopes of same. Using methods described hereinbefore, polyclonal, monoclonal antibodies, or chimeric monoclonal antibodies can be raised to a polypeptide which comprises, mimics, or cross-reacts with a B cell or T cell epitope of the polypeptide ofM pneumoniae set forth in SEQ ID NO:1 or SEQ ID NO:2 or a polypeptide ofM. genitalium set forth in SEQ ID NO: 4 or SEQ ID NO: 5 or a derivative, homologue or analogue thereof.

According to this embodiment of the present invention, an antibody molecule which binds to a polypeptide immunogen which comprises, mimics, or cross-reacts with a B cell or T cell epitope of a polypeptide of M pneumoniae or M. genitalium according to the embodiments described herein, is contacted with a second polypeptide, preferably comprising a sequence of amino acids which is a subset of, or overlaps the amino acid sequence of the polypeptide against which the antibody was raised. Standard ELISA or other immunoassay is used to assess the relative binding of antibody to the derivative polypeptide molecule. Thus, a continuous and highly immunogenic B cell or T cell epitope may be identified as the smallest amino acid sequence present in both the first polypeptide and one or more derivative polypeptides, which is highly immunoreactive with said antibody. A polypeptide molecule may comprise more than one continuous B cell or T cell epitope. A highly immunogenic, discontinuous B cell or T cell epitope may thus be inferred as any possible combination of one or more continuous B cell or T cell epitopes of a M. pneumoniae polypeptide as hereinbefore described, in particular the M.

pneumoniae polypeptide set forth in SEQ ID NO: 1 or SEQ ID NO:2 or the M genitalium polypeptide set forth in SEQ ID NO: 4 or SEQ ID NO: 5. According to this embodiment of the present invention, a discontinuous B cell or T cell epitope shall be taken to include a polymer of B cell or T cell epitopes, as hereinbefore described.

A wide range of immunoassay techniques are available as can be seen by reference to US Patent Nos. 4,016,043, 4,424,279 and 4,018,653. These, of course, include both single-site SUBSTITUTE SHEET (RULE 26) VO0 97/21727 PCT/AU96/00803 29 and two-site or "sandwich" assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target.

Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody is immobilised on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time and under conditions sufficient to allow formation of an antibody-antigen complex, a second antibody specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule.

In this case, the first antibody is raised to a polypeptide immunogen which has the characteristics of a polypeptide according to the embodiments described herein.

The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of antigen.

Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent. In accordance with the present invention, the sample is one which may contain a synthetic polypeptide substantially the same as, or derived from the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2 or SEQ ID NO: 4 and SEQ ID NO: 5, or the immunogenic B cell or T cell epitopes contained therein.

In the typical forward sandwich assay, a first antibody raised against a surface polypeptide of the present invention is either covalently or passively bound to a solid surface. The SUBSTITUTE SHEET (RULE 26) WVO 97/21727 PCT/AU96/00803 solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs, microplates or microplate wells, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking, covalent binding or adsorption. The polymer-antibody complex is washed in preparation for the test sample.

An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient 2-40 minutes) and under suitable conditions 25"C) to allow binding of any antigen present in the sample to the antibody.

Following the incubation period, the reaction locus is washed and dried and incubated with a second antibody specific for a portion of the first antibody. The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the antigen.

An alternative method involves immobilising the target molecules in the biological sample and then exposing the immobilised target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detected by direct labelling with the antibody. Alternatively, a second labelled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.

By "reporter molecule" as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules radioisotopes) and chemiluminescent molecules.

In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognised, SUBSTITUTE SHEET (RULE 26) O 97/21727 PCT/AU96/00803 31 however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, P-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. Examples of suitable enzymes include alkaline phosphatase and horseradish peroxidase. It is also possible to employ fluorogenic substrates which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labelled antibody is added to the first antibody-antigen complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of antigen which was present in the sample. The term "reporter molecule" also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.

Alternately, fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope.

As in enzyme immunoassays (EIA), the fluorescent labelled antibody is allowed to bind to the first antibody-antigen complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength and the fluorescence observed indicates the presence of the antigen of interest.

Immunofluorescence and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed. It will be readily apparent to the skilled technician how to vary the above assays and all such variations are encompassed by the present invention.

SUBSTITUTE SHEET (RULE 26) WO 97/21727 PCT/AU96/00803 32 Accordingly, a further aspect of the present invention contemplates a method of detecting a polypeptide of Mycoplasma spp., in particular a polypeptide of M pneumoniae or M.

genitalium in serum, mucus, tissue extract, or other biological fluid comprising the steps of contacting said serum, mucus, tissue extract or other biological fluid to be tested with an antibody which recognises said polypeptide or a part thereof for a time and under conditions sufficient for an antibody:polypeptide complex to form and subjecting said complex to a detecting means. The latter complex may be detected by the antibody or polypeptide, preferably the antibody, having attached thereto a reporter molecule, or by addition of a second antibody labelled with a reporter molecule.

In a particularly preferred embodiment, this aspect of the present invention contemplates a method of detecting a polypeptide ofM. pneumoniae which comprises a sequence of amino acids set forth in SEQ ID NO:1 or SEQ ID NO:2 or a derivative, homologue or analogue thereof.

Accordingly, the present invention also contemplates a kit for the rapid and convenient assay for a polypeptide ofMycoplasma spp., in particular a polypeptide of M pneumoniae orM. genitalium in serum, mucus, tissue extract, or other biological fluid Those skilled in the art will be aware that the subject kit is also useful for the purpose of determining the presence of whole cells of said Mycoplasma ssp.

The kit is compartmentalized to receive several first containers adapted to contain a polypeptide according to any of the embodiments hereinbefore described or a B cell or T cell epitope thereof in recombinant or synthetic form, and several second containers adapted to contain an antibody which recognises said polypeptide or B cell or T cell epitope thereof, wherein said antibody is optionally labelled with a reporter molecule capable of producing a detectable signal as hereinbefore described. If the antibody of the second container is not labelled with a reporter molecule, then there are also provided several third containers which contain a second antibody which recognises the first antibody and is conjugated to a reporter molecule. If the reporter molecule is an enzyme, SUBSTITUTE SHEET (RULE 26) "WO 97/21727 PCT/AU96/00803 33 then several fourth containers are provided which contain a substrate molecule for said enzyme to facilitate detection of the enzyme linked to a polypeptide:antibody complex, or to a polypeptide: antibody: antibody complex when a second antibody has been used. The reporter molecule used in this kit may also be a radio-isotope, a fluorescent molecule, or bioluminescent molecule, amongst others. Optionally, the first, second, third and fourth containers of said kit may be colour-coded for ease-of use.

In an exemplified use of the subject kit, a control reaction is carried out in which the contents of the first container are contacted with the contents of the second container for a time and under conditions sufficient for an antibody: polypeptide complex to form in said first container. At the same time the sample to be tested is contacted with the contents of the second container for a time and under conditions sufficient for an antibody:polypeptide complex to form in said second container. If the antibody of the second container provided is not labelled with a reporter molecule, then the complexes produced in said first and second containers are contacted with the antibody of the third container for a time and under conditions sufficient for a tertiary polypeptide:antibody:antibody complex to form.

The polypeptide: antibody complex or polypeptide:antibody:antibody complex is then subjected to a detecting means as hereinbefore described. In analysing the results obtained using said kit, the control reaction carried out in said first container should always provide a positive result upon which to compare the results obtained in said second container which contains the test sample.

A further aspect of the present invention provides a method of assaying for the presence of antibodies against a Mycoplasma ssp. in a mammal such as a human, said method comprising contacting a biological sample from said mammal with an isolated or recombinant polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 1I or SEQ ID NO:2 or SEQ ID NO: 4 or SEQ ID NO: 5 for a time and under conditions sufficient to enable an antibody-antigen complex formation to occur.

In a preferred embodiment, said antibody-antigen complex is subsequently subjected to a detecting means.

SUBSTITUTE SHEET (RULE 26) *O 97/21727 PCT/AU96/00803 34 In one embodiment, the antibodies present in a biological sample obtained from an individual are capable of binding to one or more epitopes of a M. pneumoniae polypeptide set forth in SEQ ID NO:1 or SEQ ID NO:2 or theM genitalium sequences set forth in SEQ ID NO: 4 or SEQ ID NO: 5 or a homologue, analogue or derivative thereof.

Preferably, the antibodies present in such a biological sample are capable of binding to one or more epitopes which in their native state are localised on the surface ofMycoplasma spp. in particular M. pneumoniae or M. genitalium for example a surface epitope of the surface polypeptide which is set forth in SEQ ID NO:2.

In a more preferred embodiment, the present invention provides a method of assaying for the presence of antibodies against a Mycoplasma ssp. in a human individual, said method comprising contacting a biological sample obtained from said individual with an isolated or recombinant polypeptide as set forth in SEQ ID NO:2 for a time and under conditions sufficient to enable an antibody-antigen complex formation to occur and subjecting said antibody-antigen complex to a detecting means.

Even more particularly, the recombinant polypeptide comprises an amino acid sequence substantially the same as amino acid residues 9 to 473 of SEQ ID NO: 2 or a homologue, analogue or derivative thereof.

According to these embodiments of the invention, it will be understood in the art that a positive result will occur only when the biological sample assayed contains antibodies against said polypeptide or a derivative, homologue or analogue thereof. Those skilled in the art will be aware that such antibodies will usually have arisen as a result of infection of the individual from whom the biological sample is derived by Mycoplasma ssp., in particular M pneumoniae or M genitalium.

Any biological sample containing antibodies is sufficient for the present purposes, the only requirement being that said biological sample contains sufficient antibodies against a surface polypeptide of Mycoplasma spp., in particular a surface polypeptide of M pneumoniae or M genitalium, to enable the detection of the antibody-antigen complex.

SUBSTITUTE SHEET (RULE 26) WO 97/21727 PCT/AU96/00803 Preferably, the biological sample is selected from the list comprising blood or blood products, mucus, respiratory epithelium, tissue of the upper respiratory tract, cerebro-spinal fluid or tissue of the central nervous system, amongst others. If difficulties are obtained in detection of an antibody-antigen complex, it is possible to purify or concentrate the immunoglobulin fraction presentin said biological sample, using any one or more standard procedures known to those skilled in the relevant art, prior to using the method hereinbefore described. The present invention extends to the use of any immunoglobulin fractions, or partially-purified antibody preparation which is obtained for the purpose of detecting antibodies as described herein.

It will also be known to those skilled in the art that the polypeptide used to detect said antibodies present in a biological sample may contain amino acid sustitutions, deletions, insertions, or other modifications including the addition of enzyme molecules, radioisotopes or fluorescent tags, amongst others, which may be useful in assisting the detection of the antibody-antigen complex formed according to this aspect of the invention.

The present invention therefore extends to the use of derivatives, homologues or analogues of the subject polypeptides used in the performance of the assay described according to this aspect of the invention.

The method described herein is at least useful for the purpose of determining whether said mammal has been, at the time a biological sample was taken, infected with a microorganism belonging to the genus Mycoplasma, in particular M pneumoniae such that antibodies to said microorganism have been produced in response to infection.

Accordingly, an alternative embodiment of the present invention provides a method of detection of Mycoplasma infection in an individual, said method comprising contacting a biological sample obtained from said individual with an isolated or recombinant polypeptide as set forth in SEQ ID NO: 1 or SEQ ID NO:2 or SEQ ID NO: 4 or SEQ ID NO: 5 or a derivative, homologue or analogue thereof, for a time and under conditions sufficient to enable an antibody-antigen complex formation to occur and subjecting said antibody-antigen complex to a detecting means and wherein said method is an SUBSTITUTE SHEET (RULE 26) v/0 97/21727 PCT/AU96/00803 36 immunoassay.

According to this embodiment of the invention, said immunoassay may be an ELISA, radioimmunoassay, histochemical test or sandwich assay.

According to the methods described in this aspect of the invention, the recombinant polypeptide is immobilised on a solid substrate and the biological sample containing antibodies against a polypeptide of Mycoplasma, in particular M. pneumoniae or M.

genitalium, is brought into contact with the bound antigen. After a suitable period of incubation, for a period of time and under conditions sufficient to allow formation of an antibody-antigen complex, a second antibody which is specific for the bound antibody and labelled with a reporter molecule capable of producing a detectable signal, may be added, and the reaction mixture incubated, allowing sufficient time for the formation of an antigen-antibody-antibody complex. Any unreacted material is washed away and the presence of antibodies in the biological sample is determined by observation of a signal produced by the reporter molecule as hereinbefore defined. Variations to the method described are numerous and will be apparent to those skilled in the art. The present invention extends to all variations of the method described herein.

Accordingly, the present invention also contemplates a kit for the rapid and convenient assay of infection by Mycoplasma spp., in particular M. pneumoniae in an individual comprising, in a first compartment several first containers adapted to contain the isolated polypeptide set forth in SEQ ID NO: 1 or SEQ ID NO:2 or SEQ ID NO: 4 or SEQ ID NO: or a derivative, homologue or analogue thereof in recombinant or synthetic form and optionally adsorbed thereto, and several second containers adapted to contain an antibody which recognises said polypeptide or a B cell or T cell epitope thereof, wherein said antibody is optionally labelled with a reporter molecule capable of producing a detectable signal as hereinbefore described. There are also provided several third containers which contain a second antibody which recognises the first antibody and is conjugated to a reporter molecule. If the reporter molecule is an enzyme, then several fourth containers are provided which contain a substrate molecule for said enzyme to facilitate detection of the SUBSTITUTE SHEET (RULE 26) WOv 97/21727 PCT/AU96/00803 37 enzyme linked to a polypeptide:antibody complex, or to a polypeptide: antibody: antibody complex when a second antibody has been used. The reporter molecule used in this kit may also be a radio-isotope, a fluorescent molecule, or bioluminescent molecule, amongst others. Optionally, the first, second, third and fourth containers of said kit may be colourcoded for ease-of use.

In an alternative embodiment, the kit may be contained in a package which comprises microtitre wells in one section, in which reactions may be performed. Accordingly, in one embodiment, the microtitre wells may be the equivalent of the first compartment hereinbefore described and contain the polypeptide set forth in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO: 4 or SEQ ID NO: 5 or a derivative, homologue or analogue thereof, adsorbed thereto.

In an exemplified use of the subject kit, the contents of the first container may be bound to a microtitre well contained in the package, if not provided in a format where said contents are already adsorbed to said microtitre well, and a biological sample to be tested is added and incubated for a time and under conditions sufficient for an antigen-antibody complex to form in said microtitre well. Following a washing step to remove unbound antibodies and other unbound protein, the contents of the third container are added to the antigen-antibody complex contained in the microtitre well and the reaction allowed to proceed for a time, and under conditions sufficient to allow the formation of the tertiary antigen-antibody-antibody complex. A positive control reaction may be performed in which the contents of the second container are added to the contents of the first container for a time and under conditions suitable for the formation of an antigen-antibody complex.

If the antibody of the second container is not labelled with a reporter molecule, then the contents of the third container may be added for a time and under conditions suitable for the formation of a tertiary antigen-antibody-antibody complex to form. The tertiary antigen-antibody-antibody complexes of the control reaction and the test sample are then subjected to a detecting means. Alternatively, if the contents of the second container are labelled with a reporter molecule the antigen-antibody complex of the control reaction may be subjected directly to a detecting means. The means of detection of a secondary antigen- SUBSTITUTE SHEET (RULE 26) Wb 97/21727 PCT/AU96/00803 38 antibody or a tertiary antigen-antibody-antibody complex are numerous, as hereinbefore described and will be known to those skilled in the art. Where said means is an enzyme reaction, the contents of the fourth container are added to said secondary or tertiary complex thus formed for a time and under conditions suitable to enable the enzyme reaction to occur. In analysing the results obtained using the subject kit, the control reaction should always provide a positive result for comparison to the results obtained for the test sample. A positive result is indicative of infection by Mycoplasma spp., in particular M. pneumoniae or M. genitalium.

A further aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides which encodes, or is complementary to a nucleic acid molecule which encodes a Mycoplasma spp. polypeptide according to any of the embodiments hereinbefore described.

In a related embodiment, the present invention provides an isolated nucleic acid molecule which encodes, or is complementary to a nucleic acid molecule which encodes a polypeptide which comprises, mimics, or cross-reacts with a B cell or T cell epitope of a Mycoplasma spp. polypeptide according to any of the embodiments hereinbefore described.

More particularly, in one embodiment the present invention provides an isolated nucleic acid molecule which encodes, or is complementary to a nucleic acid molecule which encodes a polypeptide or a derivative, homologue or analogue thereof which is obtainable from aM pneumoniae wherein said polypeptide has a molecular weight of approximately 110 kDa as determined by SDS/PAGE, or a predicted molecular weight of approximately 116 kDa, is a surface polypeptide and has adhesion properties.

Preferably, said nucleic acid molecule further comprises a sequence of nucleotides substantially the same as, or at least 40% similar to nucleotides 655-4071 of the sequence set forth in SEQ ID NO:3 or a complement or homologue, analogue or derivative thereof.

More preferably, said nucleic acid molecule is substantially the same as or at least similar to nucleotides 762-3851 of SEQ ID NO:3 or a complement thereof.

SUBSTITUTE SHEET (RULE 26) WO 97/21727 PCT/AU96/00803 39 In an alternative embodiment, the present invention provides a nucleic acid molecule which encodes or is complementary to a nucleic acid molecule which encodes a polypeptide or a derivative, homologue or analogue thereof obtainable from M. pneumoniae with a predicted molecular weight of approximately 16 kDa.

Preferably, said nucleic acid molecule further comprises a sequence of nucleotides substantially the same as, or at least 40% similar to nucleotides 1-761 of the sequence set forth in SEQ ID NO:3 or a complement or a homologue, analogue or derivative thereof.

More preferably, said nucleic acid molecule is substantially the same as or at least similar to nucleotides 250-654 of SEQ ID NO:3 or a complement or a homologue, analogue or derivative thereof.

In a further embodiment of the present invention, there is provided a nucleic acid molecule which comprises a sequence of nucleotides at least 40% sequence similar to the nucleotide sequence set forth in SEQ ID NO:3, or a complementary strand, or part thereof.

Preferably, the percentage similarity is at least 60-65%. More preferably, the percentage similarity is at least 70-75%. Yet still more preferably, the percentage similarity is at least 80-90%, including at least 91% or 93% or For the present purpose, "homologues" of a nucleotide sequence shall be taken to refer to an isolated nucleic acid molecule which is substantially the same as the nucleic acid molecule of the present invention or its complementary nucleotide sequence, notwithstanding the occurrence within said sequence, of one or more nucleotide substitutions, insertions, deletions, or rearrangements.

"Analogues" of a nucleotide sequence set forth herein shall be taken to refer to an isolated nucleic acid molecule which is substantially the same as a nucleic acid molecule of the present invention or its complementary nucleotide sequence, notwithstanding the occurrence of any non-nucleotide constituents not normally present in said isolated nucleic acid molecule, for example carbohydrates, radiochemicals including radionucleotides, reporter molecules such as, but not limited to DIG, alkaline SUBSTITUTE SHEET (RULE 26) WO 97/21727 PCT/AU96/00803 phosphatase or horseradish peroxidase, amongst others.

"Derivatives" of a nucleotide sequence set forth herein shall be taken to refer to any isolated nucleic acid molecule which contains significant sequence similarity to said sequence or a part thereof. Generally, the nucleotide sequence of the present invention may be subjected to mutagenesis to produce single or multiple nucleotide substitutions, deletions and/or insertions. Nucleotide insertional derivatives of the nucleotide sequence of the present invention include 5' and 3' terminal fusions as well as intra-sequence insertions of single or multiple nucleotides or nucleotide analogues. Insertional nucleotide sequence variants are those in which one or more nucleotides or nucleotide analogues are introduced into a predetermined site in the nucleotide sequence of said sequence, although random insertion is also possible with suitable screening of the resulting product being performed. Deletional variants are characterised by the removal of one or more nucleotides from the nucleotide sequence. Substitutional nucleotide variants are those in which at least one nucleotide in the sequence has been removed and a different nucleotide or nucleotide analogue inserted in its place.

In yet another embodiment, the present invention provides a nucleic acid molecule which hybridises under at least low stringency conditions, preferably under moderate stringency conditions, and more preferably under high stringency conditions, to the nucleic acid molecule set forth in SEQ ID NO:3, or to a complementary strand, or a part thereof.

For the purposes of defining the level of stringency, a low stringency is defined herein as being a hybridisation and/or a wash carried out in 6xSSC buffer, 0.1% SDS at 28"C or alternatively, in 6xSSC buffer, 0.5% SDS at 60"C. A moderate stringency is defined herein as being a hybridisation and/or a wash carried out in 2 x SSC buffer, 0.1% SDS at 65C. A high stringency is defined as being a hybridisation and/or wash carried out in 0.1% SSC buffer, 0.1% SDS. The conditions for varying the stringency of hybridisation reactions are well-known to those skilled in the art. Generally, the stringency is increased by reducing the concentration of SSC buffer, and/or increasing the concentration of SDS and/or increasing the temperature of the hybridisation and/or wash.

SUBSTITUTE SHEET (RULE 26) WO 97/21727 PCT/AU96/00803 41 Conditions for hybridisations and washes are well understood by one normally skilled in the art. For the purposes of clarification, (to parameters affecting hybridisation between nucleic acid molecules), reference is found in pages 2.10.8 to 2.10.16. of Ausubel et al.

(1987), which is incorporated herein by reference.

In yet still another embodiment, the present invention provides an isolated nucleic acid molecule which: encodes or is complementary to a sequence which encodes a Mycoplasma spp.

polypeptide with a predicted molecular weight of approximately 16 kDa or 116 kDa, preferably the M. pneumoniae polypeptide set forth in SEQ ID NO: 1 or SEQ ID NO:2; and (ii) hybridises under at least low stringency conditions, preferably under moderate stringency conditions, and more preferably under high stringency conditions, to the nucleic acid molecule set forth in SEQ ID NO:3, or to a complementary strand, or a part thereof; The genetic sequences which encode a Mycoplasma spp. polypeptide according to any of the embodiments hereinbefore described, in particular a genetic sequence which encodes or is complementary to a genetic sequence which encodes the polypeptides set forth in SEQ ID NO:1 or SEQ ID NO:2, may correspond to the naturally occurring sequence or may differ by one or more nucleotide substitutions, deletions and/or additions. Accordingly, the present invention extends to genes encoding said Mycoplasma polypeptides or derivatives, homologues or analogues thereof, or nucleic acid molecules which are at least useful as genetic probes, or primer sequences in the enzymatic or chemical synthesis of said gene, or in the generation of immunologically interactive recombinant molecules as hereinbefore described.

In a particularly preferred embodiment, the genetic sequences of the present invention are employed to identify and isolate similar genes, from any species of Mycoplasma, for example M. pneumoniae, M genitalium, or M. gallisepticum amongst others, and from other organisms.

SUBSTITUTE SHEET (RULE 26) WO 97/21727 PCT/AU96/00803 42 According to this aspect of the invention, there is provided an oligonucleotide molecule of at least 10 nucleotides, preferably at least 20 nucleotides and more preferably at least nucleotides in length capable of hybridising under low stringency conditions to part of the nucleotide sequence, or to a complement of the nucleotide sequence set forth in SEQ ID NO:3.

The present invention clearly contemplates a method for identifying a genetic sequence which is related to the sequence set forth in SEQ ID NO:3, said method comprising contacting genomic DNA, or mRNA, or cDNA, or parts, or fragments thereof, or a source thereof, with a hybridisation effective amount of a nucleic acid molecule comprising a sequence of nucleotides set forth in SEQ ID NO:3 or a derivative, homologue, analogue or complement thereof and then detecting said hybridisation.

The related genetic sequence may be in a recombinant form, in a bacterial cell, virus particle, bacteriophage particle, yeast cell, fungal cell, insect cell, animal cell, or a plant cell. Preferably, the related genetic sequence originates from a species of Mycoplasma, in particular M pneumoniae, M gallisepticum, M. penetrans, M. iowae, M. muris, M.

urealyticum, M pirum, M. imitans or M. genitalium, amongst others. In addition, the related genetic sequence may be bound to a support matrix, for example nylon, nitrocellulose, polyacrylamide, agarose, amongst others.

Preferably, the latter genetic sequence is labelled with a reporter molecule capable of giving an identifiable signal a radioisotope such as "P or 3 5 S or a biotintylated molecule).

An alternative method contemplated in the present invention involves hybridising two nucleic acid primer molecules of at least 10 nucleotides in length to a nucleic acid "template molecule", said template molecule herein defined as a "mycoplasma immunogen genetic sequence", or "mycoplasma-like immunogen genetic sequence", or a functional part thereof, or its complementary sequence. Specific nucleic acid molecule copies of said SUBSTITUTE SHEET (RULE 26) WO 97/21727 PCT/AU96/00803 43 template molecule are amplified enzymatically in a polymerase chain reaction, a technique that is well known to one skilled in the art.

Preferably, the nucleic acid primer molecules or molecule effective in hybridisation is contained in an aqueous mixture of other nucleic acid primer molecules. More preferably, the nucleic acid primer molecules are in a substantially pure form.

According to this embodiment of the present invention, the nucleic acid primer molecules are derived from opposite DNA strands of a genetic sequence of Mycoplasma sp., in particular M. pneumoniae, which encodes a polypeptide according to any of the embodiments hereinbefore described. Preferably, the nucleic acid primer molecules comprise any nucleotide sequence of at least 10 nucleotides preferably at least nucleotides, more preferably at least 50 nucleotides in length, wherein the nucleotide sequence of one primer molecule is contained within the nucleotide sequence set forth in SEQ ID NO:3 and wherein the nucleotide sequence of the other primer molecule is the complement of the nucleotide sequence set forth in SEQ ID NO:3.

The present invention also contemplates the use of degenerate inosine-containing primer molecules which encode, or are complementary to a nucleic acid sequence which encodes, an amino acid sequence which is at least 70% identical to a part of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2.

The present invention further contemplates the use of a single primer molecule as hereinbefore described in combination with a non-specific primer molecule to amplify genetic sequences related to the nucleotide sequence set forth in SEQ ID NO:3.

The mycoplasma immunogen genetic sequence or mycoplasma-like immunogen genetic sequence may be in a recombinant form, in a bacterial cell, virus particle, bacteriophage particle, fungal cell, yeast cell, insect cell, animal cell, or a plant cell. Preferably, the related genetic sequence originates from Mycoplasma spp., for example M. pneumoniae, M. genitalium, M. penetrans, M. iowae, M. muris, M. urealyticum, M pirum, M. imitans SUBSTITUTE SHEET (RULE 26) VWO 97/21727 PCT/AU96/00803 44 orM. gallisepticum amongst others. Furthermore, said genetic sequence may be in a crude cellular homogenate, or in a substantially purified form. Methods for the purification of genetic sequences from viral and cellular material are well known to a person skilled in the art.

The present invention extends to the detection of a nucleic acid molecule which encodes a polypeptide ofMycoplasmaspp., in particular a polypeptide ofM pneumoniae, wherein said polypeptide is according to any of the embodiments hereinbefore described and wherein said nucleic acid molecule is present in serum, mucus, tissue extract, or other biological fluid. In a particularly preferred embodiment, said method is directed to the detection of the nucleotide sequence set forth in SEQ ID NO:3 or its complement, or a derivative, homologue or analogue thereof. Accordingly, said method is useful for the purpose of detecting the micro-organism Mycoplasma spp., in particular M pneumoniae in said serum, mucus, tissue extract, or biological fluid.

The present invention clearly contemplates a kit for the rapid detection of the microorganism Mycoplasma spp., in particular M. pneumoniae in a biological sample, said kit being compartmentalized to contain in a first compartment, one or more nucleic acid molecules which encode, or are complementary to a nucleic acid molecule which encodes a polypeptide of Mycoplasma spp., in particular a polypeptide of M pneumoniae as hereinbefore described in embodiment. In a particularly preferred embodiment, the first compartment is adapted to contain one or more nucleic acid molecules which are substantially identical or at least 70% identical to the nucleotide sequence set forth in SEQ ID NO:3 or its complement or a derivative, homologue or analogue thereof.

The embodiments hereinbefore described do not extend to polypeptides or genetic sequences per se from M. genitalium or M. gallisepticum amongst others, however such embodiments do encompass the immunogenic properties and applications therefor of an immunogen comprising said polypeptide or encoded by said genetic sequence.

The nucleic acid molecule of the present invention is capable of being expressed in a SUBSTITUTE SHEET (RULE 26) 'WO 97/21727 PCT/AU96/00803 bacterial, yeast, animal or plant cell for the purpose of producing a polypeptide component of a vaccine composition as hereinbefore described.

Accordingly, yet still another aspect of the invention provides a genetic construct comprising a sequence ofnucleotides which encodes, or is complementary to a nucleotide sequence which encodes aMycoplasma spp. polypeptide as hereinbefore described in any embodiment, in particular the M. pneumoniae polypeptide set forth in SEQ ID NO:1 or SEQ ID NO:2, or a derivative, homologue or analogue thereof.

In an alternative embodiment, the genetic construct of the present invention comprises a sequence of nucleotides which encodes, or is complementary to a nucleotide sequence which encodes a polypeptide which comprises one or more immunogenic B cell or T cell epitopes which mimic, or cross-react, with a B cell or T cell epitope of a Mycoplasma spp.

polypeptide as hereinbefore described in any embodiment, in particular the M. pneumoniae polypeptide set forth in SEQ ID NO: 1 or SEQ ID NO:2.

In a preferred embodiment of the present invention, said genetic construct comprises a sequence ofnucleotides which is at least 40% similar to the nucleotide sequence set forth in SEQ ID NO:3, or its complementary nucleotide sequence, or a derivative, homologue or analogue thereof. More preferably, the percentage similarity to the nucleotide sequence set forth in SEQ ID NO:3 is at least 60-65%, still more preferably at least 70-75%, even still more preferably at least 80-90%, including at least 91% or 93% or Optionally, the nucleic acid molecule is operably linked to a promoter sequence, thereby regulating expression of said nucleic acid molecule in a virus particle, prokaryotic cell, or eukaryotic cell. It is understood in the art that viruses, including bacteriophage, utilise the transcriptional machinery of their host cell and thus, in order to achieve expression of a genetic construct in said virus it is necessary to use a promoter sequence that is capable of regulating expression in said host cell, whether a prokaryotic or a eukaryotic cell.

According to this embodiment of the present invention, a preferred promoter is one which SUBSTITUTE SHEET (RULE 26) O 97/21727 PCT/AU96/00803 46 is capable of expression in a eukaryotic cell, such as a fungal cell, insect cell, plant cell or an animal cell. It is known in the art that a promoter sequence is selected according to the specific purpose, for example the mode of gene regulation required. Promoters active in eukaryotic cells are numerous and described in the literature. More preferably, said promoter sequence regulates expression in a prokaryotic cell, for example the Escherichia coli lac promoter, or tac promoter sequences; amongst others. Additional promoters which are active in prokaryotic cells are also described in the literature.

The genetic construct optionally further comprises a terminator sequence. For the purposes of exemplification only, a suitable terminor sequence is the nopaline synthese gene terminator, or the octopine synthase gene terminator, amongst others.

The term "terminator" refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are 3'-non-translated DNA sequences.

In the genetic material of eukaryotic organisms, terminator sequences contain a polyadenylation signal which facilitates the addition of polyadenylated (ie. poly(A)) sequences to the 3'-end of a primary transcript. Many terminators are known and described in the literature. They may be isolated from genes of bacteria, fungi, viruses, animals and/or plants.

Reference herein to a "promoter" is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical genomic gene, for example a TATA box which may be required for accurate transcription initiation, or a CCAAT box sequence and additional regulatory elements upstream activating sequences, enhancers and silencers) which may alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner. A promoter is usually, but not necessarily, positioned upstream or of a structural gene, the expression of which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene.

In the present context, the term "promoter" is also used to describe a recombinant, synthetic SUBSTITUTE SHEET (RULE 26) Wo 97/21727 PCT/AU96/00803 47 or fusion molecule, or derivative which confers, activates or enhances the expression of a nucleic acid molecule which encodes, or is complementary to a nucleic acid molecule which encodes the immunogenic polypeptides of the present invention. Preferred promoters may contain additional copies of one or more specific regulatory elements, to further enhance expression and/or to alter the spatial expression and/or temporal expression of the said nucleic acid molecule. For example, regulatory elements which confer copper inducibility may be placed adjacent to a heterologous promoter sequence, thereby conferring copper inducibility on the expression of said nucleic acid molecule.

Placing a nucleic acid molecule under the regulatory control of a promoter sequence means positioning the said molecule such that expression is controlled by the promoter sequence.

Promoters are generally positioned 5' (upstream) to the genes that they control. In the construction of heterologous promoter/structural gene combinations it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function. Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, the genes from which it is derived. Again, as is known in the art, some variation in this distance can also occur.

Examples of promoters suitable for use in genetic constructs of the present invention include viral, fungal, bacterial, animal and plant derived promoters. The promoter may regulate the expression of the said molecule constitutively, or differentially with respect to the tissue in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, or plant pathogens, or metal ions, amongst others.

The genetic constructs of the present invention are particularly useful for the production of the polypeptide immunogen component of a vaccine composition, as hereinbefore SUBSTITUTE SHEET (RULE 26) SVO 97/21727 PCT/AU96/00803 48 described.

According to this embodiment of the present invention, a recombinant DNA molecule encoding an immunogenic polypeptide of the present invention as hereinbefore described in any embodiment, and/or a genetic construct comprising the same, may be introduced into a bacterial, fungal, plant, or animal cell producing a "transgenic organism", by various techniques known to those skilled in the art. The technique used for a given organism or specific type of tissue depends on the known successful techniques. Means for introducing recombinant DNA into a cell include, but are not limited to, transformation (Paszkowski et al., 1984), electroporation (Fromm et al., 1985), or microinjection of the DNA (Crossway et al, 1986), or specifically where said cell is a plant cell, by T-DNA-mediated transfer from Agrobacterium to the plant tissue.

Once introduced into a cell, the expression of the introduced gene may be assayed in a transient expression system, or it may be determined after selection for stable integration within the host genome.

A still further aspect of the present invention extends to a transgenic organism such as a plant, or a mammal, carrying the genetic constructs described herein. The present invention further extends to the progeny of said transgenic organism.

For the purposes of exemplification only, the present invention is further described by the following Figures and Examples.

In the Figures: Figure 1 is a photographic representation of an SDS/polyacrylamide gel of M. pneumoniae proteins partitioned using Triton X-114. Lane 1, molecular weight protein markers; lane 2, Triton X-114 detergent phase polypeptides; lane 3, Triton X-114 aqueous phase polypeptides; lane 4, whole cell proteins. Arrows indicate the 110 kDa Mycoplasma polypeptide (upper arrow) and a 70 kDaMycoplasma polypeptide (lower arrow enriched SUBSTITUTE SHEET (RULE 26) WO 97/21727 PCT/AU96/00803 49 in the detergent phase.

Figure 2 is a photographic representation of a western blot ofM pneumoniae polypeptides probed with 10 different human sera (lanes a-1) obtained from patients infected with M.

pneumoniae.

Figure 3 is a photographic representation of a western blot ofM. pneumoniae proteins, following trypsin-digestion of intact M pneumoniae. The blot was probed with antisera raised against the 110 kDa polypeptide of M. pneumoniae.

Figure 4 is a photographic representation of a western blot of whole cell lysates obtained from immuno-positive clones expressing the M. pneumoniae 110 kDa polypeptide. The number at the top of each lane refers to the clone number, Mr, molecular weight marker.

The blot was probed with antisera raised against the 110 kDa (116 kDa) polypeptide of M pneumoniae.

Figure 5 is a schematic representation of the EcoR1 fragment comprising the open reading frames encoding 16 kDa and 116 kDaM pneumoniae polypeptides, showing the positions of the consensus Shine-Dalgaro sequence (AAGAGCT), consensus prolipoprotein signal peptidase II cleavage site (FASLSFKLISC), Sau3AI and EcoRl cleavage sites.

Above the representation of the EcoRl fragment is a schematic representation showing the aligned Sau3A1 fragments used to produce the expression vectors pGEX 3XMPFP4, pGEX 3XMPFP3, pGEX INMP10, pGEX 3XMPFP2, pGEX 1NMP3 and pGEX 3XMP661.

Figure 6 is a graphical representation of a hydropathy plot of the 116 kDa M. pneumoniae polypeptide.

Figure 7 is a photographic representation of a Southern blot showing the presence of homologues of the EcoR1 fragment of M pneumoniae in other species of Mycoplasma.

SUBSTITUTE SHEET (RULE 26) mm WO 97/21727 PCT/AU96/00803 Species names are indicated at the top of each lane. Fragment lengths (bp) are indicated on the left of the photograph. BgII and EcoRI designate restriction enzymes used to digest the genomic DNA samples derived from each species.

SUBSTITUTE SHEET (RULE 26) WO 97/21727 PCT/AU96/00803 51 EXAMPLE 1 Strains Reference herein to Mycoplasma shall be taken to refer to M. pneumoniae strain FH grown in SP4 medium in glass bottles at 37"C.

EXAMPLE 2 Triton X-114 partitioning ofM. pneumoniae cellular proteins Triton X- 14 (Tx- 14) partitioning adapted from the method of Bordier (1981) was used to isolate amphiphilic Mycoplasma proteins in the detergent phase. Triton X-114 was precondensed three times with PBS. The culture ofM pneumoniae in a volume of 700ml was centrifuged to pellet the cells and the cell pellet washed twice with PBS. The cell pellet was resuspended in 5 ml ice cold 0.05% Tx-114 in PBS, vortexed and incubated on ice for 60 minutes. This solution was then centrifuged at 11000xg at 4"C for min. The supematant was layered on 1 ml ice cold 6% sucrose, 0.06% Tx- 114 in PBS and incubated at 37"C for 9 minutes followed by a low speed spin at 37 0

C.

The supernatant containing water soluble proteins was aspirated to a separate tube and precipitated detergent resuspended in 2 ml cold PBS.

The detergent phase enriched from amphiphilic proteins was methanol-chloroform precipitated essentially according to Wessel and Flugge (1984). The dried protein pellet was resuspended in 4M urea PBS and examined by SDS PAGE on a 10% (w/v) polyacrylamide gel, followed by Coomassie blue staining of proteins contained therein.

The most abundantM pneumoniae amphiphilic protein was seen as bands at 110-116 kDa (Figure 1).

SUBSTITUTE SHEET (RULE 26) ,WO 97/21727 PCT/AU96/00803 52 EXAMPLE 3 Western blotting with sera from humans infected with M. pneumoniae M pneumoniae whole cell and amphiphilic protein preparations were separated by SDS PAGE, transferred to PVDF membrane and Western blotted by the method described by Jacobs et al (1986). A panel of ten human sera positive for anti M. pneumoniae IgG were used as probes. All the human sera reacted with the 116 kDa protein with 80% of the sera reacting with the 116 kDa protein as the most potent immunogen in the amphiphilic protein preparations. A human sera negative for anti M pneumoniae did not bind any protein in the amphipilic preparation (not shown). Whole cell M pneumoniae preparations were probed with four human sera all of which reacted with the 116 kDa protein as the most potent immunogen (Figure 2).

EXAMPLE 4 Immunisation of rabbits M. pneumoniae amphiphilic protein preparations were electrophoresed on 10% SDS polyacrylamide gels, the 110 kDa and 70 kDa bands were excised, fragmented by grinding between glass plates and used to immunise rabbits according to Harlow and Lane (1988).

Twenty gg of protein was used per immunisation. The initial dose of antigen was delivered in Freunds complete adjuvant, subsequent doses were administered in Freunds incomplete adjuvant.

The resultant anti-110 kDa hyperimmune sera, hereinafter referred to as "rall10", was shown by western blotting to be reactive with the 110 kDa protein and to a far lesser extent with three other low molecular weight proteins, which may be proteolytic degradation products of same.

SUBSTITUTE SHEET (RULE 26) '0 WO 97/21727 PCT/AU96/00803 53 EXAMPLE The M pneumoniae 110 kDa polypeptide is a surface polypeptide AdherentM pneumoniae from 50 ml of culture were rinsed 3 times with PBS, scraped into ml PBS and divided into 8 ml aliquots. The washed cells were incubated at 37"C with 600 pg trypsin for either 15 or 30 minutes, a control was incubated without trypsin for minutes. Following the incubation, 400 gg ofsoyabean trypsin inhibitor was added to each tube. The tubes were centrifuged at 14,000xg, 4*C for 30 minutes and the cell pellets washed, centrifuged again and resuspended in 50 pl PBS. Each suspension (25 pL) was examined by western blot analysis, probed with rall0. This analysis revealed the digestion of the 110 kDa polypeptide from the surface of intact cells in the presence of trypsin (Figure The 116 kDa polypeptide was digested further to yield a 45 kDa polypeptide when the incubation period was increased to 15 min or 30 min (Figure 3).

EXAMPLE 6 DNA extraction M pneumoniae was grown in 700 ml SP4 medium in glass bottles. When the medium turned orange, the cells were harvested by scraping adherent cells into medium and centrifuging for 30' at 14,000 g, 4"C. The cell pellet was then washed twice with PBS.

DNA was extracted from the cells according to Su et al (1988).

EXAMPLE 7 Sau3A Ipartial digest ofM. pneumoniae DNA M. pneumoniae DNA was digested with a series of dilutions of the restriction enzyme Sau3A I, essentially according to Sambrook et al (1989). The digested DNA was then examined by electrophoresis on a 1.2% agarose gel. The dilution series determined SUBSTITUTE SHEET (RULE 26) .1 WO 97/21727 PCT/AU96/00803 54 that the optimal conditions for partial DNA digestion generating an average size fragment of 1500 bp was 0.035 U Sau3A I/1 ug DNA.

EXAMPLE 8 Removal of DNA fragments less than 100 bp by chromatography Pharmacia MicroSpin Sephacryl S-400 HR columns were used to remove DNA fragments smaller than 100 bp from the Sau3A I partial-digested DNA preparation. Sau3A I digested M. pneumoniae DNA (10g DNA/501l sample volume) was applied to a column and fragments larger than 100 bp were eluted in sterile dH 2

O.

The eluate was extracted with phenol:chloroform [1:1 and ethanol precipitated. The resulting DNA pellet was resuspended in 20pli sterile dH 2

O.

EXAMPLE 9 Preparation of cloning vector Amrad vector pGEX-IN was employed for the expression cloning of M pneumoniae Sau3A I digested DNA. In addition to an ampicillin resistance gene, the principal features of pGEX-IN are a lac I" mutant repressor (synthesised at 10x normal rate so that no expression of fusion protein occurs in absence of inducer), a tac promoter (hybrid trp-lac promoter), an ORF coding for the 26 kDa glutathione S-transferase from Schistosoma japonicum and a multiple cloning site (Smith and Johnson, 1988). This arrangement allows the expression of fusion proteins that may subsequently be purified by affinity interaction with Amrad Glutathione Sepharose 4B.

pGEX-1N DNA (10 pg) was digested with 40 Units of BamH I. The digested vector (6 tg) was treated with Bacterial Alkaline Phosphatase (BAP) obtained from GIBCO BRL and used according to the manufacturer's instructions, to remove 5' phosphates from same and SUBSTITUTE SHEET (RULE 26) WO 97/21727 PCT/AU96/00803 thus prevent intramolecular ligation of the vector from occurring.

The BAP treated vector was extracted twice with phenol:chloroform ethanol precipitated and resuspended in sterile dH20 to a concentration of 27 ng/pl.

EXAMPLE Ligation of M. pneumoniae Sau 3Alfragments to pGEX-lN vector DNA Ligations were performed using T4 DNA ligase (Boehringer Mannheim) 1U per ligation.

Size-fractionated Sau3A I-digested M. pneumoniae DNA (0.5 was ligated to 0.1 jg pGEX-IN, using standard procedures known in the art.

1/10 of the total ligation was used to transform electrocompetent Escherichia coli strain DH5a by electroporation using the BioRad Gene Pulser apparatus. Transformed bacteria were grown with shaking at 37"C to facilitate expression of ampicillin resistance prior to spreading on LB plates containing 50 gg/ml ampicillin for overnight growth at 37"C.

EXAMPLE 11 Immunoscreen of expression library Bacterial colonies were overlaid with Hybond C extra supported nitrocellulose membrane filters (Amersham). Filter lifts from plates were placed colony side up on fresh LB plates containing 100 ig/ml ampicillin and 2mM IPTG and incubated at 37"C for 3 hours to induce expression of fusion proteins. Cell lysis, and protein fixing were performed according to Sambrook et al (1989).

Bacterial colonies were screened for expression of fusion protein, essentially according to Jacobs et al (1986), by probing with affinity-purified ral 10 antisera. The ral 10 antisera was affinity-purified by an adaption of the method of Beall and Mitchell (1986). Briefly, SUBSTITUTE SHEET (RULE 26) WO 97/21727 PCT/AU96/00803 56 M. pneumoniae amphipilic protein preparations were transferred to PVDF membrane. The region of the membrane to which the 110 kDa protein had bound was excised, incubated with the antisera and washed. The antisera were then eluted with low pH glycine (i.e.

0.15M NaCI, 0.1M glycine pH 2.6) and neutralized immediately with 2M Tris/HC1 pH The eluted antisera did not cross-react with E. coli proteins, nor did it cross react with other M. pneumoniae proteins. Positive colonies were found at a frequency of about 1/500.

Positive colonies were grown in SOC broth with 50 pg/ml ampicillin and subsequently plated out on LB plates with 50 pg/ml ampicillin. Single positive colonies were then patched onto duplicate gridded plates, one of which was then treated and screened with antisera as described previously. The resulting eleven positive clones were picked off the duplicate plate and grown in broth. Cultures were stored as glycerol stocks as described by Sambrook et al. (1989).

EXAMPLE 12 Characterisation of immuno-positive clones DNA insert size was ascertained by digestion of CTAB plasmid minipreps with EcoR I and Sma I. Fusion protein expression was examined by SDS PAGE followed by western blotting of induced clones with ral 10 antisera (Figure For this purpose, the anti E. coli specificity of ral 10 was adsorbed to filter lifts of E. coli prior to use, as described by Sambrook et al (1989).

Immuno-positive clones were characterised further, by determining the size of the DNA inserts contained therein, and the size of internal EcoR I fragments (Table 3).

SUBSTITUTE SHEET (RULE 26) WO 97/21727 PCT/AU96/00803 57 Table 4 Sizes of restriction enzyme digested M. pneumoniae genomic DNA fragments that hybridised to radiolabelled expression clones.

Clone Number 1 2 11 12 3 8 9 Mr of expressed fusion protein (kDa) 74 74 74 74 51 90 49 31 M. pneumoniae DNA insert size (kbp) 2.9 2.4 3.6 2.8 0.7 3.9 2 0.15 Size of EcoR I fragments (kbp) 0.22, 3.3 0.14, 0.525 3.6, 0.6 0.15 EXAMPLE 13 Sequencing of expression clones The clones listed in Table 3 were sequenced using Phamacia "Sequencing kit or Deaza G/A "Sequencing Mixes as per instructions. Electrophoresis was performed with AT Biochem Long Ranger Gel in a Base Runner apparatus.

Analysis of sequence data suggested that clone 10 was the most 3' of the clones yet contained no stop codon. Clones 1, 2, 8, 9 and 12 shared identical sequence 3' to a Sau3A I site but 5' of this site had no homology indicating that ligation of Sau3A I digestion products had occurred prior to ligation with the vector. It was subsequently decided to clone the entire gene encoding the 110 kDa surface polypeptide, to facilitate sequencing of its 5' and 3' ends.

SUBSTITUTE SHEET (RULE 26) WO 97/21727 PCT/AU96/00803 58 Nucleotide sequence analysis showed that clone 8 was chimaeric containing genetic sequences from a gene unrelated to the 110 kDa polypeptide gene), since nucleotides 1-101 of clone 8 were identical to the M. pneumoniae His t-RNA gene.

EXAMPLE 14 Southern blots ofM. pneumoniae genomic DNA probed with expression clones DNA (150 ng) from clones 1,3,9,10 and 11 was radiolabelled with 20 aCi a3p-dCTP by random primed labelling (Boehringer Mannheim). Bgl II and EcoR I were used to digest 10 gg of genomic DNA from M. pneumoniae, M. gallisepticum strains Ts 11 and 6/85 and M. synoviae strains BC and 7NS. These digests were run on a 0.7% (w/v) agarose gel and transferred to nylon membrane (Amersham Hybond as per the manufacturers instructions. Digested genomic DNA from these Mycoplasma spp. was hybridised to each of the labelled clones as described by Sambrook et al. (1989) after the method of Southern (1971). Following hybridisation, membranes were then washed for 60 minutes 3 times in 0.1 x SSC at As shown in Table 4, several M. pneumoniae genomic DNA fragments hybridised to the different clones. In contrast, clones from M. pneumoniae did not hybridize to DNA from M gallisepticum or M synoviae.

The 5' ends of clones 1 and 11 were suspected to be chimaeric clones. This explanation accounts for the hybridisation of clone 1 to a Bgl II fragment other than the 10,269 bp Bgl II fragment, and also the hybridisation of clones 1 and 11 to EcoR I fragments other than the 7,874 bp and 3,295 bp Eco RI fragments. Alignment of homologous sequence from the different clones in conjunction with analysis of the Southern blot data allowed the construction of a map locating the expression clones on the gene for the 110 kDa protein and supported this conclusion.

SUBSTITUTE SHEET (RULE 26) WO 97/21727 PCT/AU96/00803 59 Table 4 Sizes of restriction enzyme digested M. pneumoniae genomic DNA fragments that hybridised to radiolabelled expression clones.

Clone Size of hybridizing Bgl II fragments (bp) 10,269 9,124 10,269 10,269 10,269 10,269 Size of hybridizing EcoR I fragments (bp) 20,647 7,874 3,295 7,874 3,295 3,295 7,874 7,874 3,295 2,464 #9 #11 EXAMPLE Cloning ofM. pneumoniae EcoR Ifragments of the 110 kDa surface polypeptide gene Synthesis of probe Analysis of the 1325 bp sequence obtained from clone 8 revealed an Nhe I restriction site at nucleotide 244 unique not only for this sequence but also for the 850 bp of noncontiguous clones 3 and 10. pGEX-IN does not contain an Nhe I site so digestion of expression clone 8 with Sma I (unique for pGEX-1N and 3' to BamH I in the multi cloning site) and the Nhe I excised a 3062 bp fragment of the M pneumoniae DNA insert.

SUBSTITUTE SHEET (RULE 26) f !WO 97/21727 PCT/AU96/00803 The 3062 bp fragment was excised from an agarose gel and purified with Prepagene (BioRad). The fragment (36 ng) was radioactively labelled as described in the preceding Examples.

The radiolabelled probe was hybridised to one of the Southern blots described previously. Consistent with data presented in Table 4, the 3062 bp probe hybridized to EcoR I fragments of 7,874 bp and 3,295 bp and to a single Bgl II fragment of 10,269 bp in M pneumoniae DNA. Thus, the probe was specific for the 110 kDa protein gene and was suitable for use in screening libraries to obtain additional clones.

II Construction of library M. pneumoniae genomic DNA (10 g) was digested with 50 U EcoR I, and applied to a Pharmacia MicroSpin Sephacryl S-400 HR column in a total volume of 50 l1, to remove fragments smaller than 100 bp. The eluted DNA was extracted with phenol:chloroform [1:1 ethanol precipitated and resuspended in sterile dH 2

O.

Plasmid pUCBM20 (Boehringer Mannheim) was cut with EcoR I and treated with Bacterial Alkaline Phosphatase followed by two extractions using phenol:chloroform [1:1 ethanol precipitation and resuspension in sterile dH 2

O.

The EcoR I digested, 5' dephosphorylated pUCBM20 was ligated to EcoR I digested M pneumoniae DNA at a vector:insert ration of 1:3, in a total volume of 9 pl.

Electrocompetent E. coli strain DH5a were transformed with 1 pl of the ligation reaction and plated as for the pGEX-1N cloning.

The plates were overlaid with Hybond N (Amersham) filters and filters subsequently prepared for hybridisation according to the manufacturers instructions. Screening of the library with radiolabelled DNA probe was as described by Sambrook et al (1989).

SUBSTITUTE SHEET (RULE 26) WO 97/21727 PCT/AU96/00803 61 Positive colonies were selected and streaked on LB plates with 50 tg/ml ampicillin.

Following overnight growth individual colonies were picked, patched onto gridded LB plates with ampicillin and grown overnight. Filter lifts were taken of the patched clones and screened as previously. Positive clones were picked and grown in SOC broth with ampicillin overnight for CTAB plasmid miniprep analysis.

Agarose gel electrophoresis ofEcoR I digested miniprep DNA samples from the positive clones allowed identification of clones containing either the 7,874 bp or 3,295 bp EcoR I fragments spanning the M pneumoniae 110 kDa protein gene.

These clones were subsequently shown to contain DNA sequence beyond the 5' and 3' limits of the clones isolated from the Sau3A I M. pneumoniae pGEX-1N expression library.

EXAMPLE 16 Sequence analysis of the 16 kDa and 116 kDa proteins No ribosomal binding site (RBS) or promoter could be identified for the ORF encoding the 116 kDa protein. However, the ORF encoding the 16 kDa protein was preceded by a consensus Shine-Dalgarno sequence (AGGAGCU) commencing at nucleotide position 239 of SEQ ID NO: 3. These data suggest that the linked ORF's encoding the 16 kDa and 116 kDa proteins are part of the same operon.

Analysis of the derived amino acid sequence of the 16 kDa polypeptide ofM.

pneumoniae revealed a consensus for a prokaryotic prolipoprotein signal sequence cleaved by signal peptidase II: FASLSFKLIEC with the cysteine at amino acid position 31 of SEQ ID NO: 1 (Figure The PSORT program, predicted the 16 kDa protein to be a bacterial membrane protein.

SUBSTITUTE SHEET (RULE 26) 4WO 97/21727 PCT/AU96/00803 62 EXAMPLE 17 The NH2 terminal half of the 116 kDa protein is highly antigenic The translation of the ORF coding for the 116 kDa protein (SEQ ID NO: 2) indicated a hydrophobic peak for the amino terminal 26 amino acids with a mean Kyte Doolittle hydrophobicity value of 162. This value was markedly higher than those for the penultimate hydrophobic peak, 75 and the entire 116 kDa protein, -31. As no consensus signal peptidase cleavage site could be detected following the leader sequence, it is probable that this region of relatively high hydrophobicity is involved in membrane association.

EXAMPLE 18 Production of derivatives of the 116 kDa polypeptide GST fusion proteins were prepared comprising various regions derived from the 116 dDa M pneumoniae polypeptide. Fusion proteins were produced by subcloning Sau3Al fragments of the open reading frame encoding the 116 kDa polypeptide into the pGEX expression vector, to produce an in-frame fusion with the GST-encoding region of the vector (Figure Positions of fusion proteins within the 116 kDa M.

pneumoniae polypeptide are indicated in Table Antisera were prepared against the GST fusion proteins using standard procedures.

EXAMPLE 19 Ability of antisera against fusion proteins to detect M. pneumoniae Thirty four serum samples from patients with suspected M pneumoniae infection were SUBSTITUTE SHEET (RULE 26) WO 97/21727 PCT/AU96/00803 63 Table Position of fusion proteins within the 116 kDa polypeptide ofM. pneumoniae Fusion protein 661 3C FP2 FP3 FP4 Nucleotide position 25-1422 1399-2127 2125-2550 2536-2689 2659-2886 2451-3087 Amino acids (SEQ ID No:2) 9-473 467-709 709-850 846-896 887-962 969-1029 969-1029 assessed for anti M. pneumoniae antibodies by the Serodia-mycoll particle agglutination assay; 33 were positive. These sera were used at a dilution of 1/600 in Western blot to assess IgG reactivity with 5 purified GST fusion proteins derived from the 116 kDa protein. Results are presented in Table 6. The fusion protein 661, containing the uncleaved signal sequence, reacted with 29 of the 34 sera Fusion protein reacted with 13 sera and fusion protein 10C and fusion protein FP3. Only one serum unreactive with fusion protein 661 reacted with another fusion protein, FP3. One serum was reactive with the GST negative control The reactivity of fusion protein 661 with sera from humans infected with M pneumoniae suggests potential in serodiagnosis. Other serum samples will be assessed by the more sensitive technique of ELISA.

EXAMPLE Other closely related mycoplasma lack homologous genes The possibility of ORF's homologous to the M pneumoniae 16 kDa and 116 kDa SUBSTITUTE SHEET (RULE 26) WO 97/21727 PCT/AU96/00803 64 ORF's in the species of the M. pneumoniae group and the phylogenetically closely TABLE 6 Detection ofM. pneumoniae in patient samples using various antisera Recombinant Vector Fusion Protein Sera from infected patients positive for IgG pGEX 3XMPFP3 FP3 32 pGEX 1NMP0I 10C 38 pGEX 3XMPFP2 FP2 18 pGEX 1NMP3 3C 24 pGEX 3XMP661 661 related M penetrans and M iowae was investigated by Southern blots, only M.

pneumoniae and M. genitalium DNA hybridised to the probes.

Whole cell proteins were analysed by Western blot for antigenic cross reactivity with the 116 kDa protein ofM pneumoniae. Only proteins derived from M. pneumoniae reacted with the monospecific rabbit anti 116 kDa. The antigenicity and specificity of the 116 kDa protein warrant further investigation of its potential as a specific and sensitive serodiagnostic reagent.

EXAMPLE 21 Isolation of a homologue of the 16 kDa encoding and 116 kDa encoding open reading frames The genomic sequence of M genitalium Fraser et al. (1995) contains contiguous open reading frames corresponding to the 16 kDa (MG074) and 116 kDa (MG075) reading frames ofM pneumoniae. The M genitalium ORF MG074 has 58.4% nucleotide SUBSTITUTE SHEET (RULE 26) WO 97/21727 PCT/AU96/00803 identity and 37.3% amino acid identity to the gene for the 16 kDa protein. TheM.

genitalium ORF MG075 has 61% nucleotide identity and 52% amino acid identity to the gene for the 116 kDa protein ofM. pneumoniae. Neither of the M. genitalium ORF's have been assigned a function although on the basis of this work they can be described as surface proteins.

MG074 and MG075 are adjacent, in the same order and on the same strand of the chromosome as the ORF's encoding the 16 kDa and 116 kDa proteins.

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

SUBSTITUTE SHEET (RULE 26) WO 97/21727 PCT/AU96/00803 66

REFERENCES

1. Ausubel, F.M. et aL (1987) In: Current Protocols in Molecular Biology, Wiley Interscience (ISBN 047140338).

2. Beall, and G.F. Mitchell 1986. J. Immunol. Methods. 86:217-223.

3. Bordier, C. 1981. J. Biol. Chem. 257:1604-1607.

4. Cimolai, N. et at 1989. J. Rheumatol. 16:1150-2.

Cimolai, et al. 1992. Microbiollmmunol. 36:465-78.

6. Cole et at (1985) In Monoclonal antibodies in cancer therapy, Alan R. Bliss Inc., pp 77-96; 7. Crossway et al. (1986) Mol. Gen. Genet. 202: 179-185; 8. Fraser, et al Science. 270: 397-403.

9. Fromm et al (1985) Proc. Natl. Acad Sci. (USA) 82:5824-5828; Goodman et al (1987) Biopolymers 26: 525-532; 11. Granstrom, T. et al JMedMicrobiol. 40:288-92.

12. Harlow, and D. Lane (1988). In:Antibodies a Laboratory Manual, Cold Spring Harbor Laboratory.

13. Huse et al. (1989) Science 246: 1275-1281; 14. Jacobs, et al (1986). Journal of Clinical Microbiology. 23:517-522.

15. Koskiniemi, M (1993). CNS manifestations associated with Mycoplasma pneumoniae infections: summary of cases at the University of Helsinki and review. Clin Infect Dis.

16. Kohler and Milstein (1975) Nature, 256: 495-499; 17. Kozbor et al. (1983) Immunol. Today 4: 72; 18. Mierke et al. (1990) Int. J. Peptide Protein Research, 35:35-45; 19. Paszkowski et al. (1984) EMBO J. 3:2717-2722; Portoghese et al. (1990) J. Med Chem. 33:1714-1720; 21. Sambrook, E.F. Fritsch, and T. Maniatis 1989. Molecular Cloning: A Laboratory Manual, second. Cold Spring Harbor Laboratory Press.

22. Smith, and K.S. Johnson 1988. Gene. 67:31-40.

23. Southern, E.M. 1975. Journal ofMolecular Biology. 98:503.

SUBSTITUTE SHEET (RULE 26) .WO 97/21727 PTA9/00 PCT/AU96/00803 67 24. Su, C..J.,et al 1988. Infect Immun. 56:3157-6 1.

Wessel, and U.I. Flugge 1984. Analytical Biochemistry. 138: 141-143.

26. Yayoshi, et al 1992. Mficrobiol Immunol. 36:455-64.

27. Zagamni, A.S. et al ,Detection of Aycoplasma pneumoniae in CSF of a patient with encephalitis. In: Australian Society for Microbiology Annual Scientific Meeting, 1994, Melbourne, Victoria, Australia: Australian Society for Microbiology.

SUBSTITUTE SHEET (RULE 26) WO 97/21727 PCT/AU96/00803 68 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: The University of Melbourne (ii) TITLE OF INVENTION: Recombinant Vaccines (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Davies Collison Cave Patent Attorneys STREET: 1, Little Collins Street CITY: Melbourne STATE: Victoria COUNTRY: Australia ZIP: 3001 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: PCT International FILING DATE: 13-DEC-1996 (vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: AU PN7127/95 FILING DATE: 13-DEC-1995 (viii) ATTORNEY/AGENT INFORMATION: NAME: Hughes, E. John L.

(ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 613 9254 2777 TELEFAX: 613 9254 2770 SUBSTITUTE SHEET (RULE 26) WO 97/21727 W097/1727PCT/AU96/00803 INFORMATION FOR SEQ ID NO:i: Wj SEQUENCE CHARACTERISTICS: LENGTH: 135 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (Xi) SEQUENCE Met Arg Lys Leu Ile DESCRIPTION: SEQ ID NO:i: Lys Leu Aen Val Ile Val Phe Val i0 Leu Gly Giu Leu Phe Ala Ser Leu Ser Phe Lys Leu Ile Leu Leu Tyr 1s Ser Cys Leu Leu Phe Val Lys Leu Ala Lys Thr Arg Phe Val Asn so Ser Ser Val Asn Gin Tyr Ser Leu Asn Gly Tyr Tyr Ile Ile Gin Lys Met Ala Asn Ser Phe 55 65 Phe Val Leu Phe Giu Thr Glu Ile Phe Leu Val Thr Asp Thr Lys Asn Lys Arg GiU Giu Gin Val 105 Giu Pro Val Arg Gin Gin His Val Ser Thr Tyr Phe Asn 115 Thr Phe Phe 130 Gin Giu Tyr His Thr Leu Asp Lys Leu Ser Tyr Phe Arg Leu Phe Lye Asp Lys 125 Phe Giu Ile SUBSTITUTE SHEET (RULE 26) WO 97/21727 W097/1727PCT/AU96/00803 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 1030 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE Met Lys Leu Ser Ala 1 5 DESCRIPTION: SEQ ID NO:2: Ile Ile Ser Lau Ser Vai Ala Gly Thr Thr Ala Val Val Pro Thr Thr Leu Val Thr Val Gly Asn Lys Thz- Asp Ile Arg Ala Ala Ala His Gin Val His Glu Ser Ser Asp Phe Phe Gly s0 Thr Arg Ser Ser Leu Aen Ser Val Pro Lys Aia Ile Thr Val Giy Thr Asp Leu Val Asn Tyr Pro Gin Leu Phe Leu Ala Lye Aen Ala Leu Leu Gin Gly Aia Leu Thr Pro Asn Gin 130 Asp Lys Phe Ser Asp Ala Lys Lys Asp Lye Ala Phe Pro Leu Phe Asn Asn Pha Thr Asp Leu Gin Val Asp Gin Ser Phe Phe Ser Ala Pro Leu 145 Asn Ser Ser Asp Lye Ser Leu Asp Phe Arg Phe Phe Thr Giu Gin Leu Leu Pro GiU Gly SUBSTITUTE SHEET (RULE 26) WO 97/21727 W097/1727PCT/AU96/00803 Val Val Val Ala Gin Lys 195 Asp Ser Lys Gin Ser Phe 180 185 Asp Tyr Tyr Val Aen Ala Ser 190 Tyr Thr Leu Leu Leu Val Leu Leu Ser Leu Pro Gly Leu 210 Asn Tyr Met Phe His Ile Thr Leu Gly Lys Val Val Asn 225 Lys Phe Ser Phe Pro Phe Lys Thr Leu Asn Leu Ala Ser Asn Val Tyr Asn 245 Gly Lys 260 Gly Val Asp Val Giu Ala Gin Lys Asn Leu 255 Val Gly Lys Lys Lye Asp 275 Tyr Leu Asn His Val Lye Ala Giu Asp Val 270 Ile Ala Lys Val Aen Ala Asn Lys Asn Gin Phe Ile Ile 290 Ala Giu Leu Met Lye Ala Leu Lye Phe Gly Aen Gin Giu Gly Gin Pro Ser Phe Leu Lys Vai Met 315 Asp Lys Val Giu Asp Phe Giu Leu Phe Asn Leu Arg Pro Gly Leu Gly Lye 335 Phe Vai Lye Thr Val Tyr 355 Leu Ile Gin Ser Ser Gin Ala Glu Aen Lye Ile 350 Leu Aen Leu Lye Leu Ile Phe Aen Lys Lys Thr Leu Lye 370 Giu Leu Ser Ile Giu Leu Asn Ser Leu Gly Leu Val Asp Val Leu Phe Asp 385 Ile Thr Asp Ser Gly Leu Tyr Giu Arg SUBSTITUTE SHEET (RULE 26) WO 97/21727 W097/1727PCTIAU96/00803 Leu Gin Ser Phe Asp Leu Ile Val Pro Ala 410 Glu Glu Lys Thr Ala Ser Pro Leu Val Lys Thr Asn Glu 415 Leu Leu Thr Gin Lys 430 Lys Gly Ile Leu Thr Asp Thr Tyr Val Phe Asp Leu 435 Asn Leu Leu Lys Aen Phe Leu 450 455 Lys His Asp Phe Gin Thr Pro Phe SUBSTITUTE SHEET (RULE 26) WO 97/21727 W097/1727PCT/AU96/00803 Ala Asp Gin Val Ile Phe Thr Giu Leu Phe 475 Asp Asn Giu Ala Phe Asp Leu Gly Giu Ala Asp Phe Vai Asp Lys Ile 490 Ala Giu 495 Leu Phe Leu Lys Asp Ser 515 Lys Arg Thr Val Asn Gly Giu Lys Ile Glu Thr 510 Gly Glu Lys Leu Leu Vai Thr Leu Lys Ser Leu Val Ala 530 Ala Leu Asp Asp Leu Asp Ser Tyr Phe Lys Aen Giu Leu Leu Asn Arg Ser 545 Lys Gly Ala Thr Asp Giu Val Ala Lys Giu Ala Lys Asp Tyr Lys Lys Glu Gin Ala Lys Ala Leu Lys Lys 570 575 Leu Phe Lys Ile Gly Giu Asn Thr Leu Ser Lys Thr 585 Asn Leu Asp 590 Giu Leu Giu Lys Ile Thr 595 Leu Lys Giu Val Asn Thr Glu Aen Giu Thr 610 Glu Thr Thr Leu Val Lye Lys Leu Vai Giu Tyr Lys Giu Leu Gly Asn Glu Ile Lys Aen Leu Ile Lys Ala Leu Giu Phe Leu Asp Pro Lye Asp Giu Thr Thr Leu Asp Lys 655 Leu Leu Phe Lye Gly Giu Ser Tyr 660 Ala Met Lye Asp Lys Tyr Ile 670 Val Pro Gly Lys Glu Gly Phe 675 Pro Gly Tyr Trp Ala Lye Gly SUBSTITUTE SHEET (RULE 26) WO 97/21727 WO 9721727PCT/ALJ96/00803 Ala Phe 690 Lys Ser Giu Ser Ile Giu Thr Phe Lys Ser Ile Axg Asp Gly Asp Met Al a 700 Phe Leu 705 Ser Ser Val Lys Gin 725 Ile Lys Tyr Gly Asp Ser Phe Ile Asp Lye Thr Gly Aen Asp Len 720 Gly Gly Ser Phe 735 Pro Ala Tyr Tyr 750 Gly Val Asp 'lhr 765 Asn Lys Giu Tyr Asp Gly Gin Asn Val Leu Ser Leu Ile Asn 755 Thr Ile Asp Ala 770 Ser Gin Ile Thr Lye Val 775 Pro Ala Ile Gin Ile Ile Val Giu Leu Lye 785 Phe Gly Lys Scr Aen Gly Gin Val 795 Met Phe Asp Ser 810 Phe Asn Val Asn Ile Leu Ser Gin Ser 800 Thr Lye Gin Ile 815 Phe Gin Lye Asn Giu Asp 835 Gin A±-g Val Tyr Gin Phe Lys Aen Ile Gin Val Thr Ser Arg Len Asp Ile Phe Ala Arg 830 Asp Pro Gin Ile Gin Leu Ile Pro Phe Val Asp Gly 850 Lye 865 Lye Ala Val Asp Lye Aen Ile Thr Lys Gin Ala 870 875 Gin LeU Tyr Gin Ala Leu 890 Asn Thr Gin Scr Pro Val Aen Lys Gin Gin Lye Gin Gin Ser Lye Gin Ser Lys Gin Len Asp Thr Lye Thr 910 SUBSTITUTE SHEET (RULE 26) 19 1 WO 97/21727 WO 9721727PCT/AU96/00803 Gin Leu Gly Tyr Leu Leu Lys Leu 915 920 Gly Asp Asn Trp Lys Asp Asp Tyr Lys 930 Ser Leu Ile Asp Asp Thr 935 Ile Ile Asn Asn Asn Tyr Leu Giu 940 Ala Ser Phe Asn Ser 945 Asp Leu Trp Leu Phe 965 Ile Thr Val Asp Leu Gly Ile Pro Lye Ile Trp Pro Phe Asn Leu 'Siu Ile Pro 975 Met Gin Gly Ser Leu Gin Leu Tyr 980 Ser Ser Val Ile Phe Pro Tyr 990 Gly Ile Tyr 995 Asp Thr Ser Val Gin Asp Ala Thr Lys Ile Val Lys Arg 1000 1005 Leu Asn 1010 Phe Thr Asp Met Giy Phe Lys Leu Asn Asp Pro Lys Pro Asn 1015 1020 Phe Trp Phe Val Gly Phe 1025 1030 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 4071 base pairs TYPE: nucieic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Vi) ORIGINAL SOURCE: ORGANISM: Mycopiasma (ix) FEATURE: NAME/KEY: CDS pneumoniae SUBSTITUTE SHEET (RULE 26) WO 97/21727 WO 9721727PCT/AU96/00803 LOCATION: 250. .654 (ix) FEATURE: NAM'E/KEY: CDS LOCATION: 762. .3851 OTHER INFORMATION: /codon- (seg: "tga" 3648. .3651, aa: Trp) /codon=(seq: "Itga" 3663. .3665, aa:-Trp) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GCACCATCCA AATGGCTTAC AACGAACAAC ACCACAAAAC ACCAATGACG GTGCAAAAAC CAATTACCTT AAACCAGCCG TTAAGTTAAA GACCAAAAGC TCAGAGCAGC AAAAAGCTGC 120 TTTAATTAAG CAGCTAACCA AGGAAATGAA GCAAGCAGCC GCTAACCAAA ACTATGAGTT 180 AGCGATTGAG ATCCGCGACT CGATCTTTGA ATTGGAAAAG CAATTTCGTG GTAAAATTAA 240 GAGCTAGCA ATG; CGT AAA CTA ATT AAA TTA AAC GTC ATT GTC TTT GTC 288 Met Arg Lys Leu Ile Lys Leu Asn Val Ile Val Phe Val TTG TTG TAC TTG GGC GAG CTG TTT GCC AGC CTT TCG TTC AAG TTA ATC Leu Leu Tyr Leu Gly Giu Leu Phe Ala Ser Leu Ser Phe Lys Leu Ile

AGT

Ser 30 TGC CTC AAG ACA CC Cys Leu Lys Thr Arg 35 AAC CAG TAC TCC TTA AAC GGG TAC TAT Aen Gin Tyr Ser Leu Aen Giy Tyr Tyr TTG TTT GTC TTT GTC AAC ATC ATC CAA AAG ATG GCT Leu Phe Vai Phe Val An Ile Ile Gin Lys Met Ala AAC TCT TTC CAA Asn Ser Phe Gin ATT AAC GAA TTT Ile Asn Giu Phe AAG TTA GCT TCC TCA GTT GTG TTG TTT GAA ACT GAA Lys Leu Ala Ser Ser Val Val-Leu Phe Giu Thr Giu TTA GTT CTC TTT ACT GAT ACA AAG AAT AAG CGT GAG GAG ACT GAA CCA Leu Val Leu Phe Thr Asp Thr Lys Aen Lye Arg Giu Glu Ser Giu Pro s0 85 528 WO 97/21727 WO 9721727PCT/AU96/00803 GTG CGC CAG GTG TCA ACA ACC CAA Val Arg Gin Val Ser Thr Thr Gin 100 CAA CAA CAC TAC TTT AAC CAC AAA Gin Gin His Tyr Phe Asn His Lys 110 115 GAG TAT CAC CAG GTT ACG CTC GAO Giu Tyr His Gin Val Thr Leu Asp 105 CTG AGC GAT TAO TTC CGT TTG TTT Leu Ser Asp Tyr Phe Arg Leu Phe 120 125 576 AAG GAC AAA Lys Asp Lys ACT TTC TTC TTT GAA ATT Thr Phe Phe Phe Glu Ile ATC TAGTTACTAA ATTGACCTGA 674 135 ATTGCAACCT TTCAGGCACT TTTTACTGTT AGTAACTAGT CTTTTTTOAT TTCAATATTT AAGTTTTTAA TTTAACCAAT TTTTAOT ATG AAG CTT AGT GOT ATT ATC TC Met Lys Leu Ser Ala Ile Ile Ser OTA TCA GTC GOT GGT ACT GTG GGA ACA ACT Leu Ser Val Ala Gly Thr Val Gly Thr Thr GCG GTG GTA GTA COT ACA Ala Val Val Val Pro Thr ACT ATA ACG OTT GTA AAT AAG ACC CAC CAA GTA GAA CAT GAA TCA Thr Ile Thr Leu Val Asn Lys Thr His Gin Val Giu His Giu Ser

GAA

881 CAA TOG GAT TTT CAA GAT ATT OGO TTT GOT OTT AAT AGT GTT Gin Ser Asp Phe Gin Asp Ile Arg Phe Gly Leu Asn Ser Val AAG TTG .Lys Leu COA AAA Pro Lys GCA CAG OCA GOT GOG GCA ACT AGA ATT ACC Ala Gin Pro Ala Ala Ala Thr Arg Ile Thr AAA TTA Lys Leu ACT GAT Thr Asp 65 GTC AAC TAT AAG TOO TCA CCA CAA Val Aen Tyr Lys Ser Ser Pro Gin GTG GAA AAC GG Vai Giu Asn Gly CAA CTC TTT TTA Gin Leu Phe Lau TTT GAT AAA TTC Phe Asp Lys Phe 977 1025 GOG AAG AAC GCG Ala Lys Asn Ala OTT AAG Leu Lys AAA CTC CAA GOT GAG Lys Leu Gin Gly Giu 100 1073 SUBSTITUTE SHEET (RULE 26) WO 97121727 WO 9721727PCT/AU96/00803 AGT GAT GCG AAG Ser Asp Ala Lys TTC CCA GCG CTA ACC Phe Pro Ala Leu Thr 115 GCT GAT TTA CAG Ala Asp Leu Gin 1121 1169 TGG GTT GAC CAA CAG Trp Val Asp Gin Gin 125 CTG TTT AAT CCA AAC Leu Phe Asn Pro Asn 130 CAA AGT TTC TTT Gin Ser Phe Phe ACT CCG CCC AGG TCA AAC Ser Ala Pro Arg Ser Asn 140 TTT ACC CTT TCA Phe Thr Leu Ser 145 TCT GAC AAA Ser Asp Lys AAG CCT ACT Lys Ala Ser 150 1217 TTA GAC Leu Asp TTT ATC TTC Phe Ile Phe 155 CGC TTT ACT AAC TTC ACC GAA TCC GTT CAG TTG Arg Phe Thr Asn Phe Thr Glu Ser Val Gin Leu 160 1 f;[ 1265 TTA AAA Leu Lys 170 CTA CCA GAA GCT GTA TCG GTT CTA CTT GAC TCC AAA CAA AC Leu Pro Giu Gly Val Ser Val Val Val Asp Ser Lys Gin Ser 1313 CAT TAC TAT GTC Asp Tyr Tyr Val CTG TCT TTA Leu Ser Leu CCA GAT Pro Asp 205 AAT GCT AGT GCC CAA AAA Asn Ala Ser Ala Gin Lys 190 195 TAC ACT TTG GGT TTA AAC Tyr Thr Leu Gly Leu Asn 210 AAG GTT GTC AAT AAA TTT Lys Val Val Aen Lys Phe 225 CTC GCC TTT AGC AAC CTT Leu Ala Phe 5cr Aen Val 240 TTA TTA GTT CTA CCG Leu Leu Val Leu Pro 200 TAT ATG TTT GAC CAC Tyr Met Phe Asp His 215 AGT TTT AAT CCC TTC Ser Phe Asn Pro Phe 230 1361 1409 1457 1505 ATT ACT TTA AAC GGT Ile Thr Leu Aen Cly 220 AAA ACG AAT TTA AAC Lye Thr Asn Leu Asn 235 TAC AAT Tyr Asn 245 CCC GTT CAT Gly Val Asp SUBSTITUTE SHEET (RULE 26) WO 97/21727 W09711727PCT/AU96/00803 GTG TTT Val Phe 250 GAA GCA CAA AAG Giu Ala Gin Lys TTA GTA GGT Leu Val Gly AAG GGT AAA TAC CTC AAC Lys Gly Lys Tyr Leu Asn 1553

ACC

Thr 265 CAC GTG AAG His Val Lys GCT GAA Ala Giu 270 GAC GTA AAG AAG Asp Val Lys Lys GTT AAT GCC AAC Val Asn Ala Asn 1601 AAA AAC CAA TTT Lys Asn Gin Phe GAC ATT GC Asp Ile Ala 285 AAA ATT ATC Lys Ile Ile 290 GCT GAG CTA ATG Ala Giu Leu Met GGT AAA Gly Lys 295 1649 GCC CTT AAA Ala Leu Lys TTT GGC Phe Gly PAT CAA CAA GA Asn Gin Gin GiU 305 GGT CAA CCA TTA TCC TTC Giy Gin Pro Leu Ser Phe 310 1697 CTA PAG GTA Leu Lye Val 315 TTA GTC CGT Leu Val Arg 330 AGT AGT CA Ser Ser Gin 345 PAC AAA PAG Asn Lys Lys ATG GAT AAA GTT Met Asp Lys Vai GPA GAT TTT Giu Asp Phe GPA AA Giu Lye 325 CTG TTT PAC Leu Phe Asn 1745 1793 CCT GGA TTG Pro Giy Leu GCA GPA PAC Ala Giu Aen 350 PAA TTT GTT PAG Lye Phe Vai Lye TTA ATC CPA AGT Leu Ile Gin Ser PAG ATA ACT GTC Lys Ile Thr Vai PAG TTA ATC TTT Lys Leu Ile Phe 1841 ACC ATC TTA PAC CTA CTT AAA GAG CTT TCC ATT Thr Ile Leu Aen Leu Leu Lye Giu Leu Ser Ile CCG GA Pro Giu 375 1889 TTA PAC TCT Leu Asn Ser TTA GGT Leu Gly TTA GTG GAC GTC Leu Val Asp Val 385 TTG TTT GAT Leu Phe Asp GTC ATT ACT Vai Ile Thr 390 1937 GAC TCT GAT Asp Ser Asp .395 GGT CTC TAT GA Giy Leu Tyr Glu TTG CPA TCT TTC Leu Gin Ser Phe AAA GAC TTA ATC Lys Asp Leu Ile 405 1985 SUBSTITUTE SHEET (RULE 26) WO 97/21727 W097/1727PCT/AU96/00803 GTT CCA Val Pro 410 GCA GTT AAA ACG Ala Val Lys Thr AAT GAA AAA ACC Asn Giu Lys Thr GCG GCT Ala Ala 420 TTA AGT CCA TTA Leu Ser Pro Leu 2033 ATT GAA GAG TTA TTA ACC Ile Giu Giu Leu Leu Thr CAA AAG GAT ACC TAT GTG TIT GAC TTA ATT Gin Lys Asp Thr Tyr Val Phe Asp Leu Ile 2081 CAA AAA CAC AAG GGT ATC TTG ACT AAC Gin Lys His Lys Gly Ile Leu Thr Asn 445 TTA AAG AAC TTC Leu Lys Asn Phe TTA GCT Leu Ala 455 2129 GAT TTC CAA AAA Asp Phe Gin Lys 460 TCA ACG CCG TTT Ser Thr Pro Phe GCT GAT CAA OTA Ala Asp Gin Val GCC ATC TTC Ala Ile Phe 470 2177 ACT GAG TTA Thr Giu Leu 475 TTT GAC AAC GAA Phe Asp Aen Giu GCG TTT GAT TTA TTT GGT GAG GCT Ala Phe Asp Leu Phe Gly Giu Ala 485 2225 GAC TTT Asp Phe 490 GTT GAC AAG ATT Val Asp Lys Ile GAA CTC TTC TTA Giu Leu Phe Leu AAG CGT ACT GTT Lys Arg Thr Val 2273 AAA AAT GGT GAA AAA ATT Lys Asn Gly Giu Lys Ile GAA ACT AAA GAT Giu Thr Lys Asp CTA CTG GTA ACA Leu Leu Vai Thr 2321 2369 TTA AAG AGT CTT Leu Lys Ser Leu GGG GAA AAG GTA Gly Giu Lys Val GCC TTA GAT GAT Ala Leu Asp Asp TTG TTA Leu Leu 535 GAT AGC TAC ATC TTT AAA AAT GAA Asp Ser Tyr Ile Phe Lys Asn Giu 540 CTT AAC CGC AGT Leu Asn Arg Ser GTA GAA GTG Val Giu Val 550 TAC AAA AAG Tyr Lys Lys 2417 GCT AAG OCT GAA Ala Lys Ala Giu 555 GCT AAG GAC ACT AAA GGT Ala Lye Asp Thr Lys Gly 560 GCT ACC GAT Ala Thr Asp 565 2465 SUBSTITUTE SHEET (RULE 26) WO097/21727 PCT/AU96/00803 GAA CAA GCT AAG CCG CTC AAA AAA CTC TTT Glu Gin Ala Lys Ala Leu Lys Lys Leu Phe 570 ;7r AAA CAC Lye His 580 ATT GCT GAA AAC Ile Gly Glu Aen 2513

ACT

Thr 585 TTA AGT AAA ACC Leu Ser Lys Thr CTC CAT AAA ATC Leu Asp Lys Ile TTA AAA GAA Leu Lys Glu CTT AAA Val Lys 600 AAG GTT Lys Val 615 2561 2609 AAC ACC GAA AAT Asn Thr Glu Asn CAA TTA GAA GAA ACT Glu Leu Glu Glu Thr CAA ACA ACC TTA Glu Thr Thr Leu AAA AAA CTC Lys Lys Leu AAG AAT GGC Lys Aen Gly 635 GTT GAA TAC AAG Val Giu Tyr Lys GAA CTT GCC AAC Glu Leu Gly Aen TTT GAA ATC Phe Glu Ile 630 GAC CCT AAA Asp Pro Lys 2657 2705 TTG ATT AAG CCA Leu Ile Lys Ala CTC GAA TTC TTG Leu Giu Phe Leu GAT TTA Asp Leu 650 GAA ACA ACT TTA Clu Thr Thr Leu AAA CTC TTC TTC Lys Leu Leu Phe GGC GAA AGC TAC Gly Clu Ser Tyr 2753 2801 GCG ATG AAA GAC Ala Met Lye Asp TAC ATC AAC Tyr Ile Lys GAA GGT Ciu Gly 675 TTC CCT GGT TAT Phe Pro Cly Tyr TGG GCT AAA GCA CTA Trp Ala Lye Gly Val 685 GTC CCT GGG GCC TTT CAA TCT Val Pro Cly Ala Phe GlU Ser 690 ATT GAA AAT ACT Ile Clu Aen Thr 695 2849 TTT AAG Phe Lye ACT CCT ATT Ser Ala Ile 700 GAT AAA ACC Asp Lye Thr AAC GAT TTA Asn Asp Leu 720 AAC TCC ATT CCT CAT CTC TTT GC Lye Ser Ile Arg Asp Leu Phe Ciy 705 710 ACC ACC CTT AAA GAA ACT CAT TCA Ser Ser Val Lye Giu Thr Asp Sez- 725 2897 GAC ATC CTC TTT CGT Asp Met Leu Phe Cly 715 2945 SUBSTITUTE SHEET (RULE 26) WO 97/21727 W097/1727PCT/AU96/00803 TTC ATC ACC CTA GGT GGT TCG TTC Phe Ile 730 Thr Leu Gly Gly Ser Phe 735 OCT TAC TAC Ala Tyr Tyr 750 GAC ATT AAG Asp Ile Lys TCT TTA ATT Ser Leu Ile 755 GGC GOT GAA AAT Gly Gly Giu Asn 2993 3041 CTC AAT GTG TTG CCA Leu Asn Val Leu Pro AAC AGC GAA ATT Aen Ser Glu Ile TAT CAA ATT ATT Tyr Gin Ile Ile OTT GAA TTA AAA Val Glu Leu Lys 780 GTA OAT ACC ACA Val Asp Thr Thr OAT GCA ACT AAG Asp Ala Thr Lys 3089 3137 3185 AAC AAA GAG TAT Asn Lys Olu Tyr GOT AAA TCG CCA OCT ATT AAC Gly Lys Ser Pro Ala Ile Asn 790 GOT CAG OTO Gly Gin Val 795 AAG TTO TCA CAA TCA TTC TTT AAT OTT Lye Leu Ser Gin Ser Phe Phe Asn Val 800 TOO ACA AAT ATO Trp Thr Aeri Met 805 TAC GAG TTC AAA Tyr Giu Phe Lys TTT GAC Phe Asp 810 OAT AAC Asp Asn 825 AOC ATT ACC Ser Ile Tbr AAA CAA ATC Lye Gin Ile 815 TTC CAA'AAG Phe Gin Lys 3233 ATC CAA OTG TTT OCO COA AAC OAA Ile Gin Val Phe Ala Arg Asn Oiu 830 GAC ATT TCT OAT CCT OAA CAA CG Asp Ile Ser Asp Pro Oiu Gin Arg AAC ACA TCA COT Asn Thr Ser Arg 3281 OAA CTT Oiu Leu OTA ATT CCA TTT OCT TTT Val Ile Pro Phe Ala Phe 855 3329 OTT OAT Vai Asp GOC TTT Oly Phe 860 GOC ATT CAA CTC Oly Ile Gin Leu OCA OTT GAC AAA Ala Vai Asp Lys AAC ATT ACO Aen Ile Thr 870 CAA CTT TAT Gin Leu Tyr 3377 3425 AAA GAA OCA Lye Oiu Ala 875 GOT AAT ACT GAO CCA Giy Asn Thr Oiu Pro 880 AAA TCT CCT OTA ATT Lys Ser Pro Vai Ile SUBSTITUTE SHEET (RULE 26) WO 97/21727 W097/1727PCT/AU96/00803 GAG GCA CTT AAT AAA GMA AAA GAT CAA AAA CAA CAA AGT AAA CAA TCT 3473 Giu Ala Leu Asn Lys 890 Glu Lys Asp 895 Gin Lys Gin Gin Ser Lys Gin Ser 900 CCA AAA Pro Lys 905 CAA CTT GAT Gin Leu Asp ACT AMA ACA CAG TTG Thr Lys Thr Gin Leu 910 TAC CTA TTG AAA TTA Tyr Leu Leu Lys Leu 920 3521 GGC GAC AAT TGG GlY Asp Asn Trp AMA GAT GAT Lys Asp Asp TAC AMA Tyr Lys 930 AGC TTA ATT Ser Leu Ile GAT GAT ACG Asp Asp Thr 935 AAG ATA ACG Lys Ile Thr 950 3569 3617 ATC ATC AAT Ile Ile Asn AAC TAT TTA GAG Aen Tyr Leu Giu AGC TTT PAT TCC Ser Phe Asn Ser GTT GAT Val Asp TTG GGT ATT CCT Leu Giy Ile Pro GAC CTT TGA Asp Leu Trp TTG TTT AAG ATT TGA Leu Phe Lys Ile Trp 965 3665 3713 COT MAG Pro Lys 970 TTC PAT TTG GMA Phe Asn Leu Giu CCA ATG CAA GGT Pro Met Gin Giy TTA CPA CTT TAC Leu Gin Leu Tyr AGT AGT GTT ATC TTC Ser Ser Vai Ile Phe 990 CCA TAC GGT ATT TAT GAC ACC AGT Pro Tyr Giy Ile Tyr Asp Thr Ser 995 GTT CA Vai Gin 1000 GGC TTC Giy Phe 1015 3761 GAT GCT ACG PAG ATT GTG PAG Asp Aia Thr Lys Ile Vai Lys 1005 CGT CTG PAC TTT Arg Leu Asn Phe 1010 ACT GAC ATG Thr Asp Met 3809 AMA CTC PAC GAT CCA AMA COT AMC Lys Leu Asn Asp Pro Lys Pro Asn 1020 TTC TGG TTC Phe Tip Phe 1025 GTT GGT Val Giy

TTT

Phe 1030 TAGAGCAGAC TPACTACTAT ACTGATTPAG CTACCTAGPA GGTAAAGGCG AGPAGTTCAT ccACcAAJ~c CCGCGGGTTG TTTACGGTAG GATGAACTTT TTGCTTTATA CTCGTCAAAA CTPATGTTGG GCTTGGCGTA TATCTACCTA GTTTTAGATA TTGGTTAAAC TTGCTPATTT 3851 3911 3971 4031 SUBSTITUTE SHEET (RULE 26) 1 WO 97/21727 WO 9721727PCT/AU96/00803 84 CGTACCGTGC TCCATCACAG GTCTTTAAGC GCACTATACC 4071 IN4FORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 137 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Mycopiasma genitalium (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Arg Gin Phe Ile Lys Leu Ser Leu Leu Val Phe Val Leu Leu Phe Leu Ser Giu Leu Ile Cys Arg Phe Arg Leu Vai Lys Ala Arg Phe Leu Lys Tyr Lys Ser Ser Val Ser Tyr Thr Aen Ser Ile Cys Leu Leu Lys Leu Ala Ser Phe Gin Asn Phe Ser Aen Ala Phe Aen Trp Val Phe Trp Phe Glu Asn Asp Vai Giu Leu Leu Ser Phe Tyr Phe Asn Phe Asp Gin Lys Ser Lye Val Asp Tyr Asn Phe Vai Vai Giu Lye Giu Gin 110 Phe Aen Gly Lye Val Thr Aia Leu Leu Thr 115 Cys Lye Leu Ser Tyr Tyr Arg Leu Phe Arg Asp Lys 125 SUBSTITUTE SHEET (RULE 26) WO W97/21727 PCT/AU96/00803 Thr Phe Trp Phe Giu Leu Ile Asn Asn 130 135 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1024 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Mycopiasma. genitalium Cxi) SEQUENCE DESCRIPTION: SEQ ID Met Lys Leu Ser Thr Ile Thr Thr Ile Cys Leu Ser Phe Gly Thr Thr Asn His Gin Gin Ile A3q Phe Gly Ala Ile Ala Leu Pro Thr Thr Val 25 Gin Asn Thr Giu Lys Gin Gin Asn 40 Ile Ser Gly Aia Ala Leu Leu Lys Pro Ile Lys Asp Thr Ile Pro Leu Leu Asn His Gin Thr Val Val Giu Val Gin Val Thr Asn Asn Pro Asn Lys Ala Ile Val Asp Lys Asp Aia Pro Gin Lys Phe Phe Leu Lys Ser Ala Leu Aen Asn Lys Leu Gin Val 100 Asn Ala Leu Asn 115 Giu Phe Asp Lys Leu Arg Thr Gly Val Ile 110 Thr Leu. Phe Ala Asp Leu Trp Ile Asp SUBSTITUTE SHEET (RULE 26) 0 i WO097/21727 PTA9/00 PCT/AU96/00803 Ile Pro Asn Gin 130 Ser Phe Phe Asp Leu Ser 135 Ala Asn 140 Lys Leu Aen Leu Thr 145 Leu Ser Asn Gin Ser Giu Val Ser Leu 150 Leu Giu Phe Ile Phe 160 Asp Gly 175 Thr Asn Phe Ser Asp Lys Asn Gin Pro 165 Lys Leu Pro Phe Ser Val Val Thr Leu Gin 195 Asn Ala Aen Glu Tyr Thr Tyr Ser Val Lys Aia 190 Asp His Ser Lys Leu Lys Vai Thr Tyr Ser Arg Vai Gly 210 Ile Ser Tyr Ala Pro Thr Val Ser Asn Gly Lys Thr Aen Asp Phe Ser Asn Pro Phe Lys Aen Ile Asn Phe Phe Lys Asn Val Asn Ala Leu Aen Phe Giu Ala Gin Gin Tyr 255 Leu Vai Gly Giy Lys Phe Leu Gin Lys Val Asn Ala Asp Asp 270 Val Lye Asn 275 Asp Ile Asn Asn Ile Glu Thr Gin Phe Aen Val Ala 285 Lys Ile 290 Thr Ala Thr Leu Gly Lys Ala Phe Gin Phe Gly Glu His 305 Lye Aen Gly Gin Leu Ser Leu Leu Val Leu Ser Gly Aen Asn Giu Phe Gin Leu Phe Asn Val Arg Pro Gly Leu Gly 335 Asp Phe Val Ser 340 Asp Leu Ile Gin Ser Ser Gin Ser Ser Asn Lye 350 SUBSTITUTE SHEET (RULE 26) i V WOT/97/217270 PCT/AU96/00803 Lys Thr Val Tyr- Gin Leu Leu Phe Giu Asn Lys Thr 360 Ile Ile His LeU Leu Gin Asp Leu Asn Ile 370 375 Sex- Giu Leu Asn Vai Leu Pro Vai Asp Ile Leu Phe Giy Ile Asn Sex- Giu Sex- Leu Tyr Arg Ile Gin Sex- Phe Lys Asp Leu Ile 405 Pro Aia Leu Lys Aila Asp 415 Lys Gin Leu Sex- Leu Giu Ala Ile Leu Aia Val Leu Asp Asn 430 Ser Ile Leu Pro Asn Thr Tyr Vai Phe Asp 435 Vai Tyr Gin Asn Phe Asn 450 Leu Leu Ser Asp Leu Lys Aen Thr Al a 460 Aen Thr Leu Pro Leu Gin Giu Gin Asp Ile Vai Asn Leu Phe Aia Asn Aia Ile Phe Asp Leu Phe Ser Asn Aia 485 Phe Vai Giu Lys Ile Aia 495 Asp Leu Phe Thr Lys 5cr S is Aia Lys Gin Lys Gin Giu Vai Asn Asn Asp Giy Thr Leu Lys Thr Lys Ile Vai Ser Ile Leu Val Giy Leu 530 Vai Giy Asp Gin Ser Ser Ile Thr Leu Leu Asn Ile Ile Phe Giu Aen Giu 550 Gin Thr 565 Phe Leu Asn Arg Asp Ser Asn Ser Val Lye Lye Gin Asp Ser Leu Aen Leu Phe Ser Vai Ile 575 SUBSTITUTE SHEET (RULE 26) 11 i WO 97/21727 W097/1727PCT/AU96/00803 Gly Asp Ile Leu Ser Glu Thr Asn 580 Asn Lys Ile Thr Lou His Ala 590 Val Lys Asn 595 Asn Giu Leu Leu Leu Vai Giu Thr Ser Thr Leu Lys Ile 610 Lys His Leu Asn Gin Tyr Lys Val Leu Val Asp Lys Phe 620 Lou Lys Aen Ser Ile Lys Giu Lou Aen Phe Phe Pro Thr Lye Asp Ile Pro Thr Ile Lys Val Leu Phe Giu Ser Giu 655 Asn Tyr Lys Tyr His Trp 675 Leu Arg Lys Lys Giu Aen Giu Giy Phe Pro Giy 670 Ser Ala Giu Ala Lys Phe Ile Pro Gly Thr Phe Asn Thr 690 Phe Tyr Ser Ala Asp Lye Thr Lye Ser Ile AX-g Asp Leu 700 Ser Vai Asn Asp Ser Ala Asp Met Leu Gly Lys Ser Leu Asp Ser Phe Ile Ile Am-i Gly Ser Thr Leu Lys Tyr His Gly 735 Asp Aen Leu Val Gly Tyr 755 Lou Lou Pro Asn His Ser Leu Ile Thr Lye Aen 750 Aia Arg Lou Gin Ile Val Asn Val 760 Asn Thr 775 Aen Phe His Ile Lou Thr 770 Ile Lye 785 Ala Giu Leu Gin Val Phe Ser Pro Lys Pro Vai Ser Pro Val Leu Ser Lye Ser Phe Giu Val Trp SUBSTITUTE SHEET (RULE 26) 1) WO 97/21727 WO 9721727PCT/AU96/00803 Thr Ile Phe Giu Asn 805 Phe Lys Asp Aen Leu 820 Ser Val Asn Gin Leu Lys Lys Giu Tyr Thr 815 Lys Phe Phe Phe Lys Ala Asp Giy Ser Ser 830 Ile Pro Phe Arg Leu Giu Phe 835 Asp Leu Ser Pro Asp Gin Arg Ala Phe 850 Val Asp Gly Tyr Phe Gin Leu Lye Lye Giu Leu Ile Pro 860 Lye Giu Thr Lys Giu Aia Aen Ser Pro Vai Leu Lys Tyr Asp Ala Vai Lye Arg Asn Asp Arg 885 Tyr Arg Pro Asn His His 895 His Asp Asp Arg Aen Tyr Pro Ser Leu Lys Ser Gin 905 Leu Giu Leu 910 Phe Ile Asp Ile Leu Asn 915 Leu Gly Asp Lys Lye Aia Aen Asn Asp Thr 930 Vai Vai Asn Ala Gin Tyr Lys Thr Phe Lye Ser Thr Lys Val Asn Ser Gly Ile Pro Ile Leu Phe Phe Phe Leu Trp Leu Lye Aen Leu Giu Ile Ile Asp Giy Ser Leu Thr 975 Leu Thr Ser Asn Val Vai Phe Tyr Ser Leu Arg Leu Asn Tyr Asp Thr Ser 990 Phe Thr Asp Thr 1005 Ser Asn Giu 995 Phe Thr Arg Ile Vai Asp 1000 Aen Phe Tyr Leu Lye Asp Aia Phe Pro Aen Phe Trp Phe Vai Giy Phe 1010 1015 1020 SUBSTITUTE SHEET (RULE 26)

Claims (73)

1. An isolated or recombinant immunogenic polypeptide derived from a Mycoplasma ssp. wherein said polypeptide has the properties of a surface polypeptide and a predicted molecular weight of approximately 16 kDa or 116 kDa, and comprises a sequence of amino acids having at least 70% similarity to any one of SEQ ID NOS: 1, 2, 4 or 5 or a fragment thereof.

2. The isolated or recombinant immunogenic polypeptide according to claim 1 wherein said polypeptide is derived from Mycoplasma pneumoniae.

3. The isolated or recombinant immunogenic polypeptide according to claim 2 wherein said polypeptide comprises a sequence of amino acids set forth in SEQ ID NO: 1 or which is at least 70% similar to all or a part thereof.

4. The isolated or recombinant immunogenic polypeptide according to claim 2 wherein said polypeptide is an immunogenic homologue, analogue or derivative of SEQ ID NO: 1 as hereinbefore defined.

5. The isolated or recombinant immunogenic polypeptide according to claim 2 wherein said polypeptide is a surface polypeptide which has adhesion properties.

6. The isolated or recombinant immunogenic polypeptide according to claim 5 wherein said polypeptide has a molecular weight of approximately 110 kDa as determined by SDS/PAGE or a predicted molecular weight of approximately 116 kDa. 25 7. The isolated or recombinant immunogenic polypeptide according to claim 6 wherein said polypeptide comprises an amino acid sequence substantially as set forth in SEQ ID NO: 2 or which is at least 70% similar to all or a part thereof.

8. The isolated or recombinant immunogenic polypeptide according to claim 6 wherein said polypeptide is an immunogenic homologue, analogue or derivative of SEQ ID NO: 2 as hereinbefore defined which is at least 70% identical thereto. WO 97/21727 PCT/AU96/00803 91

9. The isolated or recombinant immunogenic polypeptide according to claim 8 wherein the immunogenic derivative comprises an amino acid sequence selected from the list comprising residues 9 to 473, residues 467-709, residues 709 to 850, residues 846 to 896, residues 887 to 962 or residues 969 to 1029 of SEQ ID NO: 2. The isolated or recombinant immunogenic polypeptide according to claim 9 wherein the immunogenic derivative comprises amino acid residues 9 to 473 of SEQ ID NO: 2 or a fragment thereof comprising a B cell or T cell epitope.

11. The isolated or recombinant immunogenic polypeptide according to any one of claims 1 to 10 wherein said polypeptide is encoded by an isolated DNA molecule comprising a sequence of nucleotides substantially the same as SEQ ID NO: 3 or a homologue, analogue or derivative thereof which is at least 40% identical thereto.

12. The isolated or recombinant immunogenic polypeptide according to claim 11 wherein the percentage identity to SEQ ID NO: 3 is at least 60-65%.

13. The isolated or recombinant immunogenic polypeptide according to claim 11 wherein the percentage identity to SEQ ID NO: 3 is at least 70-75%.

14. The isolated or recombinant immunogenic polypeptide according to claim 11 wherein the percentage identity to SEQ ID NO: 3 is at least 80-90%. The isolated or recombinant immunogenic polypeptide according to claims 4 or 8 wherein the homologue is derived from a Mycoplasma ssp. selected from the list comprising M. penetrans, M. iowae, M. gallisepticum, M. genitalium, M. imitans, M. muris, M. urealyticum or M. pirum, amongst others.

16. The isolated or recombinant immunogenic polypeptide according to claim wherein the homologue is derived from M. genitalium. SUBSTITUTE SHEET (RULE 26) P:\OPER\MRO\10647-97.CLI 11/6/98 92

17. The isolated or recombinant immunogenic polypeptide according to claim 16 wherein said polypeptide comprises a sequence of amino acids which is substantially identical to SEQ ID NO: 4 or is at least 70% similar to all or a part thereof.

18. The isolated or recombinant immunogenic polypeptide according to any one of claims 1 to 4 or claims 15 to 17 wherein said polypeptide is a surface polypeptide.

19. The isolated or recombinant immunogenic polypeptide according to claim 16 wherein said polypeptide comprises an amino acid sequence substantially as set forth in SEQ ID NO: or which is at least 70% similar to all or a part thereof.

20. The isolated or recombinant immunogenic polypeptide according to any one of claims 1 to 19 when expressed in a virus particle, prokaryotic cell or eukaryotic cell. S 21. The isolated or recombinant immunogenic polypeptide according to claim 20 wherein the prokaryotic cell is a bacterial cell.

22. The isolated or recombinant immunogenic polypeptide according to claim 21 wherein the bacterial cell is a Escherichia coli cell or a Mycoplasma ssp. cell. S S S23. The isolated or recombinant immunogenic polypeptide according to any one of claims to 22 wherein said polypeptide is expressed as a fusion polypeptide.

24. The isolated or recombinant immunogenic polypeptide according to claim 23 wherein the fusion polypeptide is a fusion with glutathione-s-transferase. A vaccine composition for the therapeutic or prophylactic treatment of a mammalian subject against infection by a Mycoplasma ssp., said composition comprising the isolated or recombinant immunogenic polypeptide according to any one of claims 1 to 24 in an amount S sufficient to mediate an immune response when administered to said mammal and a P:\OPER\MRO\10647-97.CL1 11/6/98 -93- pharmaceutically acceptable carrier or diluent. 0@ *4 0O** 0O 0eO@ 0 S6 0W

26. The vaccine composition according to claim 25 wherein the mammal is a human.

27. The vaccine composition according to claim 25 or 26 wherein the means of administration is injection or ingestion.

28. The vaccine composition according to claim 25 or 26 or 27 wherein the Mycoplasma ssp. is M pneumoniae.

29. The vaccine composition according to claims 25 or 26 or 27 wherein the Mycoplasma ssp. is M genitalium. The vaccine composition according to any one of claims 25 to 29 wherein said vaccine composition is capable of inducing humoral immunity against said Mycoplasma ssp.

31. The vaccine composition according to claim 30 wherein said vaccine composition further prevents the onset, development or progression of symptoms associated with M pneumoniae infection when administered to said mammal.

32. The vaccine composition according to claim 31 wherein the symptoms associated with M pneumoniae infection are selected from the list comprising atypical pneumoniae, lung lesions and inflammatory reactions of the respiratory tract or central nervous system.

33. The vaccine composition according to any one of claims 25 to 32 further comprising an adjuvant.

34. An immunologically interactive molecule which is capable of binding to the recombinant or isolated immunogenic polypeptide according to any one of claims 1 to 24. WO 97/21 7 2 7 PCT/AU96/00803 94 The immunologically interactive molecule according to claim 34 when prepared against a polypeptide immunogen which comprises, cross-reacts or mimics a B cell or T cell epitope of the Mycoplasmapneumoniae polypeptide according to any one of claims 2 to 14 or a fusion polypeptide thereof.

36. The immunologically interactive molecule according to claim 34 when prepared against a polypeptide immunogen which comprises, cross-reacts or mimics a B cell or T cell epitope of the Mycoplasma genitalium polypeptide according to any one of claims to 19 or a fusion polypeptide thereof.

37. The immunologically interactive molecule according to any one of claims 35 to 36 wherein said molecule is an antibody molecule.

38. A method of detecting a Mycoplasma ssp. in a biological sample derived from a mammalian subject, said method comprising the steps of contacting said biological sample with the antibody molecule according to claim 37 for a time and under conditions sufficient for a complex to form between said antibody and a Mycoplasma ssp. polypeptide present in said sample and subjecting said complex to a detecting means.

39. The method according to claim 38 wherein the Mycoplasma ssp. is M. pneumoniae. The method according to claim 38 wherein the Mycoplasma ssp. is M. genitalium.

41. The method according to any one of claims 38 to 40 wherein the detecting means is a reporter molecule.

42. The method according to any one of claims 38 to 40 wherein the detecting means is a second antibody labelled with a reporter molecule. SUBSTITUTE SHEET (RULE 26) p1 11 W097/21727 PCT/AU96/00803

43. The method according to claim 41 or 42 wherein the reporter molecule is an enzyme, fluorophore, radionuclide-containing molecule, bioluminescent molecule or a chemiluminescent molecule.

44. The method according to claim 43 wherein the enzyme is selected from the list comprising horseradish peroxidase, glucose oxidase, P-galactosidase and alkaline phosphatase. The method according to any one of claims 38 to 44 wherein the biological sample is derived from serum, mucus, tissue or a fluid of said mammalian subject.

46. The method according to any one of claims 38 to 45 wherein the mammal is a human.

47. A kit for the rapid and convenient assay of Mycoplasma ssp. or a polypeptide derived therefrom in a biological sample, said kit comprising a first compartment adapted to contain the isolated or recombinant immunogenic polypeptide according to any one of claims 1 to 24 and a second compartment adapted to contain the antibody according to claim 37.

48. The kit according to claim 47 wherein said kit further comprises a third compartment adapted to contain a second antibody molecule capable of binding to the contents of the second compartment and conjugated to a reporter molecule.

49. The kit according to claim 48 wherein the reporter molecule is an enzyme. The kit according to claim 49 wherein said kit further comprises a fourth compartment adapted to contain a substrate molecule for said enzyme.

51. The kit according to any one of claims 47 to 50 wherein the Mycoplasma ssp. is M. pneumoniae. SUBSTITUTE SHEET (RULE 26) WO 97/21727 PCT/AU96/00803 96

52. The kit according to any one of claims 47 to 50 wherein the Mycoplasma ssp. is M genitalium.

53. A method of assaying for the presence of antibodies against a Mycoplasma ssp. in a mammal said method comprising contacting an antibody containing biological sample derived from said mammal with the isolated or recombinant immunogenic polypeptide according to claims 1 to 24 for a time and under conditions sufficient to enable an antibody-antigen complex to form.

54. The method according to claim 53 wherein the antibody-containing biological sample is selected from the list comprising blood or blood products, mucus, respiratory epithelium, tissue of the upper respiratory tract, cerebro-spinal fluid or tissue of the central nervous system.

55. The method according to claim 53 or 54 wherein the Mycoplasma ssp. is M. pneumoniae.

56. The method according to claim 53 or 54 wherein the Mycoplasma ssp. is M genitalium.

57. The method according to any one of claims 53 to 56 wherein the mammal is a human.

58. The method according to any one of claims 53 and 57 wherein said method comprises the step of purifying or concentrating the immunoglobulin fraction present in said antibody-containing biological sample prior to the step of contacting said sample with the isolated or recombinant immunogenic polypeptide and wherein said contacting step further employs said purified or concentrated immunoglobulin fraction in place of the untreated antibody-containing biological sample.

59. The method according to any one of claims 53 to 58 wherein said method SUBSTITUTE SHEET (RULE 26) P:\OPER\MRO\10647-97.CL1 11/6/98 97 comprises the additional step of detecting the antibody-antigen complex using a detecting means. The method according to claim 59 wherein the detecting means is a reporter molecule.

61. The method according to claim 59 wherein the detecting means is a second antibody labelled with a reporter molecule.

62. The method according to claim 60 or 61 wherein the reporter molecule is an enzyme, fluorophore, radionuclide-containing molecule, bioluminescent molecule or a chemiluminescent molecule.

63. The method according to claim 62 wherein the enzyme is selected from the list comprising horseradish peroxidase, glucose oxidase, P-galactosidase and alkaline phosphatase.

64. The method according to any one of claims 54 to 63 when used to diagnose a past or present Mycoplasma infection in a human subject.

65. The kit according to claim 47 wherein said kit further comprises a third compartment adapted to contain a second antibody capable of binding to the contents of the first compartment and conjugated to a reporter molecule.

66. The kit according to claim 65 wherein the reporter molecule is an enzyme.

67. The kit according to claim 66 wherein said kit further comprises a fourth compartment adapted to contain a substrate molecule for said enzyme.

68. The kit according to any one of claims 65 to 67 wherein the Mycoplasma ssp. is M pneumoniae. SWO 97/21727 PCT/AU96/00803 98

69. The kit according to any one of claims 65 to 67 wherein the Mycoplasma ssp. is M genitalium. An isolated nucleic acid molecule comprising a sequence of nucleotides which encodes or is complementary to a nucleic acid molecule which encodes the isolated or recombinant immunogenic polypeptide according to any one of claims 1 to 24.

71. An isolated nucleic acid molecule which comprises a sequence of nucleotides which is at least 40% identical to SEQ ID NO: 3 or a complementary strand thereto or at least contiguous nucleotides thereof.

72. The isolated nucleic acid molecule according to claim 71 wherein the percentage identity of SEQ ID NO: 3 is at least 60-65%.

73. The isolated nucleic acid molecule according to claim 71 wherein the percentage identity to SEQ ID NO: 3 is at least 70-75%.

74. The isolated nucleic acid molecule according to claim 71 wherein the percentage identity to SEQ ID NO: 3 is at least 80-90%. The isolated nucleic acid molecule according to claim 71 wherein said molecule is substantially identical to SEQ ID NO: 3 or a complementary strand thereof.

76. An isolated nucleic acid molecule capable of hybridising under at least low stringency conditions to SEQ ID NO: 3 or a complementary strand thereto or to at least contiguous nucleotides thereof.

77. The isolated nucleic acid molecule according to claim 76 wherein the stringency conditions are at least medium stringency conditions as hereinbefore defined.

78. The isolated nucleic acid molecule according to claim 76 wherein the stringency SUBSTITUTE SHEET (RULE 26) P:\OPERXMRO\10647-97.CLI 11/6/98 -99- conditions are at least high stringency conditions as hereinbefore defined.

79. The isolated nucleic acid molecule according to any one of claims 71 to 77 wherein said nucleic acid molecule further encodes the isolated or recombinant immunogenic polypeptide according to any one of claims 1 to 24. A genetic construct comprising the isolated nucleic acid molecule according to any one of claims 70 to 79 operably linked to a promoter sequence which is capable of regulating expression of said nucleic acid molecule in a virus particle, prokaryotic cell or eukaryotic cell.

81. The genetic construct according to claim 80 when used to produce the recombinant polypeptide.

82. The genetic construct according to claim 81 wherein the recombinant polypeptide is a polypeptide immunogen.

83. The genetic construct according to claim 82 wherein the polypeptide immunogen comprises an amino acid sequence substantially the same as any one of SEQ ID NOs: 1, 2, 4 or

84. The isolated or recombinant immuogenic polypeptide according to any one of claims 1 to 24 substantially as hereinbefore described with reference to the Figures and/or Examples. The immunologically interactive molecule according to any one of claims 34 to 37 substantially as hereinbefore described with reference to the Figures and/or Examples.

86. The method according to any one of claims38 to 46 substantially as hereinbefore described with reference to the Figures and/or Examples.

87. The method according to any one of claims 53 to 64 substantially as hereinbefore P:\OPER\MRO\10647-97.CLI 11/6/98 -100- described with reference to the Figures and/or Examples.

88. The isolated nucleic acid molecule according to any one of claims 70 to 79 substantially as hereinbefore described with reference to the Figures and/or Examples.

89. The genetic construct according to any one of claims 80 to 83 substantially as hereinbefore described with reference to the Figures and/or Examples. DATED this ELEVENTH day of JUNE, 1998. THE UNIVERSITY OF MELBOURNE by DAVIES COLLISON CAVE Patent Attorneys for the Applicants 0* a a 0*@e a. a. S a. a S. a a S..