# Patent

## Title
Antigen composition against mycoplasma

## Abstract

An isolated antigen against a Mycoplasma, prepared by a method including providing a sample of a Mycoplasma and an antibody probe, probing the Mycoplasma sample with the antibody probe to detect at least one antigen, and isolating the antigen detected. The antibody probe includes at least one antibody against the Mycoplasma that is produced by a method including (a) providing a biological sample taken a short time after an immune animal has been challenged with a Mycoplasma or Mycoplasma extract taken from the infection site or an area of a lesion or an area close to the infection site or lesion; (b) isolating cells from the biological sample; (c) culturing the cells in vitro in a suitable culture medium; and (d) harvesting antibodies produced from the cell.

## Background
The present invention relates to protective and diagnostic antigens, the preparation thereof, and their use in the formation of vaccine compositions, particularly vaccine compositions against Mycoplasma hyopneumoniae infections.
Mycoplasma hyopneumoniae is a ubiquitous swine respiratory pathogen causing mycoplasmal pneumoniae in swine (swine enzootic pneumonia). Swine enzootic pneumonia is probably the most widespread and economically significant disease in swine producing countries of the world. The economic effects of swine enzootic pneumonia (SEP) are complex, and the cost of the disease is severe. In Australia, the disease was estimated in 1988 to cost approximately $20,000,000 per annum. Increased mortality, decreased growth weight, depressed feed conversion, susceptibility to secondary bacterial infections, increased management costs, and increased use of antibiotics, are the main reasons for the economic impact of SEP.
Whilst several experimental vaccines have been produced, these have resulted in less than optimal results, and utilizing various classes of antibiotics such as tetracycline, lincamycin and tiamulin is still the most widespread control treatment. Such antibiotics are, however, of limited therapeutic value, because they do not prevent the establishment of an infection, and lung lesions may develop after treatment ends.
European Patent Application 359,919 to ML Technology Ventures L.P. describes a series of antigens, 36 kD, 41 kD, 74.5 kD and 96 kD in size, and proposes the use of such antigens in vaccines. Results presented suggest that some protection in pigs against challenge was achieved.
However, there remains a need in the art for an effective vaccine against M. hyopneumoniae which would confer protection against colonization and clinical disease following M. hyopneumoniae challenge and also significantly reduce the morbidity and mortality from secondary infections.
Accordingly, it is an object of the present invention to overcome, or at least alleviate, one or more of the difficulties and deficiencies in the prior art.
Accordingly, in a first aspect of the present invention there is provided a protective antigen against a Mycoplasma, preferably Mycoplasma hyopneumoniae prepared by a method including
providing

    
    
        a sample of a Mycoplasma; 
        an antibody probe including at least one antibody against a Mycoplasma produced by a method including;
        
            providing a biological sample taken a short time after an immune animal has been challenged with a Mycoplasma or Mycoplasma extract taken from the infection site or an area of a lesion or an area close to the infection site or lesion;
            isolating cells from the biological sample;
        
        
        culturing cells in vitro in a suitable culture medium; and
        harvesting antibodies produced from said cells;
    
    


probing the Mycoplasma sample with the antibody probe to detect at least, one antigen; and
isolating the antigen detected.
The protective antigens may also function as diagnostic antigens as discussed below.
Accordingly, in a preferred aspect of the present invention there is provided a putative protective antigen against Mycoplasma hyopneumoniae, or related infections, selected from antigens having approximate molecular weights of 110–114, 90–94, 72–75, 60–64, 52–54 and 46–48 kilodaltons (kD), as hereinafter described, mutants, derivatives and fragments thereof. The protective antigen may be a surface protein. The protective antigen may be a surface lipoprotein or membrane protein.
Preferably the protective antigens are selected from antigens having approximate molecular weights of 110–114, 90–94, 74, 62, 52 and 48 kD.
Preferably, the 72–75 kD antigen includes the following N-terminal amino acid sequence: (SEQ ID NO:12)

    
    
        AGXLQKNSLLEEVWYLAL

and, optionally, one or more of the following internal amino acid sequences: (SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15 respectively)

        AKNFDFAPSIQGYKKIAHEL
        NLKPEQILQLLG
        LLKAEXNKXIEEINTXLDN
    
    


Preferably, the 60–64 kD antigen includes one of the following N-terminal amino acid sequences: (SEQ ID NO:10 and SEQ ID NO:11 respectively)

    
    
        MKLAKLLKGFX(N/L)(M/V)IK
        ADP(F/I)(R/E)Y(V/A)PQG(Q/A)X(MIN)VG
    
    


Preferably, the 52–54 kD antigen includes the following N-terminal amino acid sequence: (SEQ ID NO:7)

    
    
        AGXWAKETTKEEKS

and, optionally, one or more of the following internal amino acid sequences: (SEQ ID NO:8, and SEQ ID NO:9 respectively)

        AWVTADGTVN
        AIVTADGTVNDNKPNQWVRKY.
    
    


Preferably, the 46–48 kD antigen includes the following N-terminal amino acid sequence: (SEQ ID NO:3)

    
    
        AGXGQTESGSTSDSKPQAETLKHKV

and, optionally, one or more of the following internal amino acid sequences: (SEQ ID NO:4; SEQ ID NO:5; and SEQ ID NO:6 respectively)

        TIYKPDKVLGKVAVEVLRVLIAKKNKASR
        AEQAITKLKLEGFDTQ
        KNSQNKIIDLSPEG
    
    


The 46–48 kD antigen may be encoded by a nucleic acid fragment: (SEQ ID NO:1)










        10         20         30         40         50


1234567890 1234567890 1234567890 1234567890 1234567890
















ATGAAAAAAA TGCCACTATA CCAGAGGAAA GAGCAGTATA TAAAATAATT
  50


 


AAAATTACAT TTTCTTCATT TGCGCCAGAA TTTTTAAGAA TTAGTACATT
 100


 


AAAAAGTAGA ACAAAAGTTA TTAATGTAAA CATTAGCGCA ATCCTTAAGA
 150


 


AAAAATTAAA AGTTTTATCT ATTTTTTTTA ATCGAAATCC AACCAGGCAT
 200


 


AAATCTTTGT CAGTATTTAT CAAGTCGGTA TTTTTTCATT ATTTCTACTA
 250


 


AAATATTATT TGAATTTGCA TTTTCCATAA TCTAAAATTT TACATTTTTT
 300


 


TATAACAATT TTTAAAAATT ACTCTTTAAT TTATAGTATT TTTTTATTTT
 350


 


TTAGTCTAAA TTATAAAATT ATCTTGAATT TTATTTGAAT TTTTATAATT
 400


 


TAGTACTAAA AAATACAAAT ATTTTTTCCT ATTCTAAGAA AAATTCATTT
 450


 


TTTAAAAAAA ATTGATTTTT ATAGTATAAT TTGTTTGTAT AATTGAATTA
 500


 


ACTTGATTTG AAAGGGAACA AAATGAAAAA AATGCTTAGA AAAAAATTCT
 550


 


TGTATTCATC AGCTATTTAT GCAACTTCGC TTGCATCAAT TATTGCATTT
 600


 


GTTGCAGCAG GTTGTGGACA GACAGAATCA GGTTCAACTT CTGATTCTAA
 650


 


ACCACAAGCC GAGACGCTAA AACATAAAGT AAGTAATGAT TCTATTCGAA
 700


 


TAGCACTAAC CGATCCGGAT AATCCTCGAT GAATTAGTGC CCAAAAAGAT
 750


 


ATTATTTCTT ATGTTGATGA AACAGAGGCA GCAACTTCAA CAATTACAAA
 800


 


AAACCAGGAT GCACAAAATA ACTGACTCAC TCAGCAAGCT AATTTAAGCC
 850


 


CAGCGCCAAA AGGATTTATT ATTGCCCCTG AAAATGGAAG TGGAGTTGGA
 900


 


ACTGCTGTTA ATACAATTGC TGATAAAGGA ATTCCGATTG TTGCCTATGA
 950


 


TCGACTAATT ACTGGATCTG ATAAATATGA TTGGTATGTT TCTTTTGATA
1000


 


ATGAAAAAGT TGGTGAATTA CAAGGTCTTT CACTTGCTGC GGGTCTATTA
1050


 


GGAAAAGAAG ATGGTGCTTT TGATTCAATT GATCAAATGA ATGAATATCT
1100


 


AAAATCACAT ATGCCCCAAG AGACAATTTC TTTTTATACA ATCGCGGGTT
1150


 


CCCAAGATGA TAATAATTCC CAATATTTTT ATAATGGTGC AATGAAAGTA
1200


 


CTTAAAGAAT TAATGAAAAA TTCGCAAAAT AAAATAATTG ATTTATCTCC
1250


 


TGAAGGCGAA AATGCTGTTT ATGTCCCAGG ATGAAATTAT GGAACTGCCG
1300


 


GTCAAAGAAT CCAATCTTTT CTAACAATTA ACAAAGATCC AGCAGGTGGT
1350


 


AATAAAATCA AAGCTGTTGG TTCAAAACCA GCTTCTATTT TCAAAGGATT
1400


 


TCTTGCCCCA AATGATGGAA TGGCCGAACA AGCAATCACC AAATTAAAAC
1450


 


TTGAAGGGTT TGATACCCAA AAAATCTTTG TAACTCGTCA AGATTATAAT
1500


 


GATAAAGCCA AAACTTTTAT CAAAGACGGC GATCAAAATA TGACAATTTA
1550


 


TAAACCTGAT AAAGTTTTAG GAAAAGTTGC TGTTGAAGTT CTTCGGGTTT
1600


 


TAATTGCAAA GAAAAATAAA GCATCTAGAT CAGAAGTCGA AAACGAACTA
1650


 


AAAGCAAAAC TACCAAATAT TTCATTTAAA TATGATAATC AAACATATAA
1700


 


AGTACAAGGT AAAAATATTA ATACAATTTT AGTAAGTCCA GTAATTGTTA
1750


 


CAAAAGCTAA TGTTGATAAT CCTGATGCCT AA
1782









Accordingly, in a further aspect the present invention provides an isolated nucleic acid fragment encoding a putative protective antigen against Mycoplasma hyopneumoniae or related infections, said nucleic acid fragment: (SEQ ID NO:1)










        10         20         30         40         50


1234567890 1234567890 1234567890 1234567890 1234567890
















ATGAAAAAAA TGCCACTATA CCAGAGGAAA GAGCAGTATA TAAAATAATT
  50


 


AAAATTACAT TTTCTTCATT TGCGCCAGAA TTTTTAAGAA TTAGTACATT
 100


 


AAAAAGTAGA ACAAAAGTTA TTAATGTAAA CATTAGCGCA ATCCTTAAGA
 150


 


AAAAATTAAA AGTTTTATCT ATTTTTTTTA ATCGAAATCC AACCAGGCAT
 200


 


AAATCTTTGT CAGTATTTAT CAAGTCGGTA TTTTTTCATT ATTTCTACTA
 250


 


AAATATTATT TGAATTTGCA TTTTCCATAA TCTAAAATTT TACATTTTTT
 300


 


TATAACAATT TTTAAAAATT ACTCTTTAAT TTATAGTATT TTTTTATTTT
 350


 


TTAGTCTAAA TTATAAAATT ATCTTGAATT TTATTTGAAT TTTTATAATT
 400


 


TAGTACTAAA AAATACAAAT ATTTTTTCCT ATTCTAAGAA AAATTCATTT
 450


 


TTTAAAAAAA ATTGATTTTT ATAGTATAAT TTGTTTGTAT AATTGAATTA
 500


 


ACTTGATTTG AAAGGGAACA AAATGAAAAA AATGCTTAGA AAAAAATTCT
 550


 


TGTATTCATC AGCTATTTAT GCAACTTCGC TTGCATCAAT TATTGCATTT
 600


 


GTTGCAGCAG GTTGTGGACA GACAAAATCA GGTTCAACTT CTGATTCTAA
 650


 


ACCACAAGCC GAGACGCTAA AACATAAAGT AAGTAATGAT TCTATTCGAA
 700


 


TAGCACTAAC CGATCCGGAT AATCCTCGAT GAATTAGTGC CCAAAAAGAT
 750


 


ATTATTTCTT ATGTTGATGA AACAGAGGCA GCAACTTCAA CAATTACAAA
 800


 


AAACCAGGAT GCACAAAATA ACTGACTCAC TCAGCAAGCT AATTTAAGCC
 850


 


CAGCGCCAAA AGGATTTATT ATTGCCCCTG AAAATGGAAG TGGAGTTGGA
 900


 


ACTGCTGTTA ATACAATTGC TGATAAAGGA ATTCCGATTG TTGCCTATGA
 950


 


TCGACTAATT ACTGGATCTG ATAAATATGA TTGGTATGTT TCTTTTGATA
1000


 


ATGAAAAAGT TGGTGAATTA CAAGGTCTTT CACTTGCTGC GGGTCTATTA
1050


 


GGAAAAGAAG ATGGTGCTTT TGATTCAATT GATCAAATGA ATGAATATCT
1100


 


AAAATCACAT ATGCCCCAAG AGACAATTTC TTTTTATACA ATCGCGGGTT
1150


 


CCCAAGATGA TAATAATTCC CAATATTTTT ATAATGGTGC AATGAAAGTA
1200


 


CTTAAAGAAT TAATGAAAAA TTCGCAAAAT AAAATAATTG ATTTATCTCC
1250


 


TGAAGGCGAA AATGCTGTTT ATGTCCCAGG ATGAAATTAT GGAACTGCCG
1300


 


GTCAAAGAAT CCAATCTTTT CTAACAATTA ACAAAGATCC AGCAGGTGGT
1350


 


AATAAAATCA AAGCTGTTGG TTCAAAACCA GCTTCTATTT TCAAAGGATT
1400


 


TCTTGCCCCA AATGATGGAA TGGCCGAACA AGCAATCACC AAATTAAAAC
1450


 


TTGAAGGGTT TGATACCCAA AAAATCTTTG TAACTCGTCA AGATTATAAT
1500


 


GATAAAGCCA AAACTTTTAT CAAAGACGGC GATCAAAATA TGACAATTTA
1550


 


TAAACCTGAT AAAGTTTTAG GAAAAGTTGC TGTTGAAGTT CTTCGGGTTT
1600


 


TAATTGCAAA GAAAAATAAA GCATCTAGAT CAGAAGTCGA AAACGAACTA
1650


 


AAAGCAAAAC TACCAAATAT TTCATTTAAA TATGATAATC AAACATATAA
1700


 


AGTACAAGGT AAAAATATTA ATACAATTTT AGTAAGTCCA GTAATTGTTA
1750


 


CAAAAGCTAA TGTTGATAAT CCTGATGCCT AA
1782









As cross protection between various Mycoplasma such as M. hyorhinis and M. synoviae has been documented, similar antigens may also be detected in other Mycoplasma species (FIG. 1).
In a still further aspect the present invention provides a method for preventing Mycoplasma infection in animals. Preferably the Mycoplasma disease is a Mycoplasma hyopneumoniae disease such as swine enzootic pneumonia (SEP). This method includes administering to an animal an effective amount of at least one protective antigen against Mycoplasma as described above.
The present invention further provides a vaccine composition including a prophylactically effective amount of at least one protective antigen against a Mycoplasma as herein described. Preferably the veterinary composition includes two or more protective antigens as herein described.
Accordingly in a preferred aspect the present invention provides a vaccine composition including two or more protective antigens selected from antigens having approximate molecular weights of 110–114, 90–94, 72–75, 60–64, 52–54 and 46–48 kilodaltons.
The vaccine composition may include any combination of two or more protective antigens selected from antigens having approximate molecular weights of 110–114, 90–94, 72–75, 60–64, 52–54 and 46–48 kD. The two or more antigens may be selected from antigens falling within one of the specified approximate molecular weights and/or antigens from different specified approximate molecular weights. The composition may contain 3, 4, 5 or 6 antigens selected from protective antigens having molecular weights of approximately 110–114, 90–94, 72–75, 60–64, 52–54 and 46–48 kD.
The vaccine compositions according to the present invention may be administered orally or may be administered parenterally (for example by intramuscular, subcutaneous, intradermal or intravenous injection). The amount required will vary with the antigenicity of the active ingredient and need only be an amount sufficient to induce an immune response typical of existing vaccines.
Reactive experimentation will easily establish the required amount. Typical initial doses of vaccine or veterinary compositions may be approximately 0.001–1 mg active ingredient/kg body weight. The dose rate may increase or multiple doses may be used as needed to provide the desired level of protection.
The vaccine composition according to the present invention may further include a veterinary acceptable carrier, diluent or excipient therefor. Preferably the active ingredient may be suspended or dissolved in a carrier. The carrier may be any solid or solvent that is nontoxic to the animal and compatible with the active ingredient. Suitable carriers include liquid carriers, such as normal saline and other nontoxic salts at or near physiological concentrations, and solid carriers, such as talc or sucrose.
Preferably the vaccine contains an adjuvant, such as Freund's adjuvant, complete or incomplete, or immunomodulators such as cytokines may be added to enhance the antigenicity of the antigen if desired.
More preferably the adjuvant is of the mineral-oil type as these have been found to be consistently superior at inducing antibody titers and Delayed Type Hypersensitivity responses. A particularly preferred adjuvant is that marketed under the trade designation Montanide ISA-50 and available from Seppic, Paris, France.
When used for administering via the bronchial tubes, the vaccine is suitably present in the form of an aerosol.
In a still further aspect of the present invention there is provided a diagnostic kit including a diagnostic antigen against a Mycoplasma, preferably Mycoplasma hyopneumoniae, identified and purified as described above.
The protective antigens according to the present invention may be isolated and identified utilizing the general methods described in Australian patent application 49035/90, the entire disclosure of which is incorporated herein by reference.
Accordingly, in a further aspect, the present invention provides a method for producing at least one antibody against a Mycoplasma. This method includes
providing a biological sample taken a short time after an immune animal has been challenged with a Mycoplasma or Mycoplasma extract taken from the infection site or an area of a lesion or an area close to the infection site or lesion;
isolating cells from the biological sample;
culturing cells in vitro in a suitable culture medium; and
harvesting antibodies produced from said cells.
The Mycoplasma may be Mycoplasma hyopneumoniae. 
The animal may be a mammal including humans. The mammal may be a domestic animal such as a pig, sheep or cattle.
The biological animal sample may be of any suitable type. The biological sample may be taken from animal tissue, organs, lymph or lymph nodes. The biological sample may be taken from the infection site, the lungs of the animal, or an area of a lesion which may be formed or an area close to the infected site or a lesion such as in the lymph nodes draining from the lungs.
However, serum/plasma samples are not used as the biological samples according to this aspect of the present invention. It has been found that the majority of antibodies found in a serum/plasma sample are irrelevant to protection or specific diagnosis or a Mycoplasma or are unrelated to the Mycoplasma. In addition, other serum/plasma components may interfere with the specific reactions between pathogen components and antibodies to them.
In contrast, the probes described in the present invention are highly enriched in Mycoplasma-specific antibodies of particular importance to protective immunity.
It is preferred that the biological samples are taken from the animals at a predetermined time in the development of the disease. In general, for a Mycoplasma infection, it has been found that the biological samples should be taken approximately 2 to 7 days after challenge with or after administration of products obtained from a pathogen or with the pathogen itself.
The cells isolated from the biological sample may include B cells.
Thus, preferably the cells are taken a short time after in vivo stimulation, preferably within approximately 2 to 5 days thereafter, resulting in the in vivo induction of antibody forming cells which will secrete specific antibodies into the culture medium after in vitro incubation.
In vitro secretion of antibodies in the culture medium by recently activated B cells may be enhanced by the addition of helper factors to the cultures. The helper factors may be cytokines used alone or in combination, including Interleukin 1, 2, 3, 4, 5, 6, 7 and 8, colony stimulating factors, interferons and any other factors that may be shown to have an enhancing effect on specific B cell secretion.
The method of producing an antibody may include a further step of activating the cells isolated to proliferate and secrete and/or release antibodies.
The cell activation step may include adding a cell activating agent to the culture medium. The cell activating agent may be selected from mitogens and helper factors produced by leukocytes, or their synthetic equivalents or combinations thereof.
The mitogens may be selected from products derived from pokeweed (Phytolacca americana) also known as pokeweed mitogen (PWM), polyvinylpyrrolidone (PVP), polyadenylic-polyuridylic acid (poly(A-U)), purified protein derivate (PPD), polyinosinic-polycytidilic acid (poly(I-C)), lipopolysaccharide (LPS), staphylococcal organisms or products thereof, Bacto-streptolysin O reagent (SLO), Staphylococcal phage lysate (SPL), Epstein-Barr virus (EBV), Nocardia water-soluble mitogen (NWEM), phytohemagglutinin (PHA), Concanavalin A (Con A), and dextran-sulphate and mixtures thereof. The cell proliferation agent may be any agent that indirectly or directly results in B cell proliferation and/or antibody secretion such as solid-phase anti-immunoglobulin. The helper factors may be cytokines including interleukin 1, 2, 3, 4, 5, 6, 7 and 8, colony stimulating factors, interferons and any other helper factors that may be shown when added alone, or in combination with other factors and agents, to have an enhancing effect on specific B cell proliferation and/or antibody secretion. This in no way is meant to be an exhaustive list of mitogens and cell actuating agents including helper factors.
The in vitro culturing of the cells may be conducted with or without prior steps to separate sub-populations of cells. The harvesting of antibodies may be conducted by harvesting of the supernatant from the culture medium. This supernatant contains antibodies secreted by these cells during the in vitro culture or artificially released from the B cells, for example by lysis of the B cells. It has been found that the antibody-containing supernatants may be used directly to detect antigens of the Mycoplasma. 
In a preferred aspect of the present invention, there is provided a method for identifying an antigen associated with a Mycoplasma, preferably Mycoplasma hyopneumoniae. This method includes
providing

    
    
        a sample of a Mycoplasma; and
        an antibody probe including at least one antibody against a Mycoplasma; 
    
    


probing the Mycoplasma sample with the antibody probe to detect at least one antigen; and
isolating the antigen detected.
The sample of Mycoplasma may be mixed with a standard buffer solution and placed on a standard support such as an SDS-polyacrylamide gel to separate the proteins contained thereon (FIG. 2).
Alternatively, the proteins may be selected utilizing the non-ionic detergent Triton X-114 (TX-114). Insoluble material may be removed by centrifugation. Proteins soluble in the TX-114 phase may then be precipitated out (FIG. 2).
The separate proteins may then be transferred to nitrocellulose, nylon or other sheets.
The probing with a suitable antibody may further include subjecting the product produced thereby to a detection assay. The detection assay may include Western blot techniques. The detection assay may be an immunoprecipitation assay, a radioimmunoassay, an enzyme-linked immunoassay or immunofluorescent assay (FIGS. 3, 4 and 5).
The antibody produced as described above may be utilized simply in the form of the supernatant harvested from the culture medium. Alternatively, the antibodies may be separated and purified.
In a further preferred aspect of the present invention the antibody contained in the culture medium may be used for the affinity purification, preferably immuno-affinity purification of antigen.
Accordingly, in a preferred aspect there is provided a method for purifying antigen. This method includes
providing

    
    
        a crude antigen mixture; and
        an antibody against a Mycoplasma immobilized on a suitable support;
    
    


subjecting the crude antigen mixture to affinity chromatography utilizing the immobilized antibody; and
isolating the purified antigen so formed.
The antibody is produced by the method described above.
Antibody can be obtained from the culture supernatant probe by conventional methods. For example, methods usually used to purify immunoglobulins from serum or plasma, e.g. precipitation with ammonium sulphate, fractionation with caprylic acid, ion exchange chromatography, or by binding and elution from immobilized protein G or protein A, may be utilized. Antibody so obtained can then be coupled to suitable supports, e.g., CNBr-activated Sepharose 4B (Pharmacia), Affi-gel (Bio-RAD), or other affinity chromatography supports able to bind proteins.
Immobilized antibody can then be applied to the fractionation and purification of specific antigen from a complex Mycoplasma extract by affinity chromatography. After binding of antigen to immobilized antibody, unbound macromolecular species can be washed away from the solid support with, e.g. buffers containing 1.5 M NaCl. Subsequently the antigen can be eluted from the affinity column with, e.g. low or high pH buffer or buffers containing chaotropic ions, e.g. 0.5–3.0 M sodium thiocyanate.
The application of the antibody probe to affinity chromatography enables sufficient quantities of specific antigens to be rapidly isolated from a complex crude extraction mixture for biochemical characterization, amino-acid sequencing and vaccination of animal for limited protection studies. Application of affinity chromatography for obtaining antigen(s) avoids the difficulties often encountered when applying conventional biochemical techniques to the purification of an antigen about which little or no data is known. It also obviates the need to raise polyclonal or monoclonal antibodies for the purpose of “analytical” affinity chromatography. Large scale preparation may, however, require the preparation of polyclonal or monoclonal antibodies.
Having identified the antigen(s) molecular biology, chemical techniques, e.g. cloning techniques, may be used to produce unlimited amounts of this antigen or, alternatively, synthetic peptides corresponding to different fragments of the identified antigens may be used as a means to produce a vaccine.
Accordingly in a preferred aspect of the present invention there is provided a method for preparing a synthetic antigenic polypeptide against Mycoplasma, preferably Mycoplasma hyopneumoniae, which method includes
providing

    
    
        a cDNA library or genomic library derived from a sample of Mycoplasma; and
        an antibody probe as described above;
    
    


generating synthetic polypeptides from the cDNA library or genomic library;
probing the synthetic polypeptides with the antibody probe; and
isolating the synthetic antigenic polypeptide detected thereby.
Either cDNA or genomic libraries may be used. The cDNA or genomic libraries may be assembled into suitable expression vectors that will enable transcription and the subsequent expression of the clone cDNA, either in prokaryotic hosts (e.g. bacteria) or eukaryotic hosts (e.g. mammalian cells). The probes may preferably be selected from

    
    
        (i) synthetic oligonucleotide probes based on the amino acid sequence of the antigen identified and purified as described above;
        (ii) antibodies obtained from the culture medium produced as described above;
        (iii) monoclonal or polyclonal antibodies produced against the antigens identified and purified as described above;
        (iv) recombinant or synthetic monoclonal antibodies or polypeptides with specificity for the antigen, e.g. as described by Ward et al., Nature, 241, pages 544–546 (1989).
    
    


The synthetic antigenic polypeptide produced in accordance with the invention may be a fusion protein containing the synthetic antigenic peptide and another protein.
In a further aspect of the present invention there is provided a DNA fragment encoding a putative protective antigen against Mycoplasma or related infections, said DNA fragments having a nucleic acid sequence according to FIGS. 6a and 6b or an homologous sequence and functionally active fragments thereof.
In a further preferred aspect of the present invention there is provided a clone including a DNA fragment encoding a putative protective antigen against Mycoplasma or related infections, said DNA fragments having a nucleic acid sequence according to FIGS. 6a and 6b or an homologous sequence and functionally active fragments thereof.
Preferably the clone is pC1-2.
The present invention will now be more fully described with reference to the accompanying Examples and drawings. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.



IN THE FIGURES
FIG. 1: SDS-Polyacrylamide gel (12.5%) profiles of SDS extracts of species of Mycoplasma—Coomassie R250 stained.
















Lane 1
Pre-stained Molecular Weight Standards



Lane 2
M. gellisepticum



Lane 3
M. synoviae.



Lane 4
M. hyopneumoniae.



Lane 5
M. hyorhinis.



Lane 6
M. flocculare.










FIG. 2: SDS-Polyacrylamide gel (12.5%) profiles of extracts of strains of M. hyopneumoniae—Coomassie R250 stained gel













Lane 1
Pre-stained Molecular Weight Standards.


Lane 2
Triton X-114 extract of M. hyopneumoniae - strain Beaufort.


Lane 3
As for Lane 2.


Lane 4
SDS extract of M. hyopneumoniae strain Beaufort.


Lane 5
SDS extract of M. hyopneumoniae strain 10110.









FIG. 3: Western blots of Triton X-114 extracted antigens from M. hyopneumoniae strain Beaufort, probed with serum and supernatant antibody probes.
















Lane 1
No antibody control



Lane 2
Dookie pig serum control 1/200.



Lane 3
Pig 105 supernatant.



Lane 4
Pig 1 supernatant.



Lane 5
Dookie pig supernatant.










FIG. 4: Western blots of SDS extracted antigens from M. hyopneumoniae strain Beaufort probed with paired serum and supernatantantibody probes. Fractionation of antigens on SDS Polyacrylamide gel (12.5%).
















Lane 1
a) Pig 453 supernatant.




b) Pig 453 serum 1/100.



Lane 2
a) Pig 105 supernatant.




b) Pig 105 serum 1/100.



Lane 3
a) Pig 1 supernatant.




b) Pig 1 serum1/100.



Lane 4
a) Pig 15 supernatant.




b) Pig 15 serum 1/100.



Lane 5
a) Dookie supernatant.




b) Dookie serum 1/100.



Lane 6
No antibody control.










FIG. 5: Western blots of SDS extracted antigens from M. hyopneumoniae strain Beaufort probed with paired serum and supernatantantibody probes. Fractionation of antigens on SDS Polyacrylamide gel (10.0%).
















Lane 1
a) Pig 453 supernatant.




b) Pig 453 serum 1/100.



Lane 2
a) Pig 105 supernatant.




b) Pig 105 serum 1/100.



Lane 3
a) Pig 1 supernatant.




b) Pig 1 serum 1/100.



Lane 4
a) Pig 15 supernatant.




b) Pig 15 serum 1/100.



Lane 5
a) Dookie supernatant.




b) Dookie serum 1/100.



Lane 6
No antibody control.










FIG. 6: The entire 48 k gene sequence (SEQ ID NO:1).
FIG. 7: The 48 kDa protein sequence of the 48 k gene sequence (SEQ ID NO:2).



EXAMPLE 1
Mycoplasma Hyopneumoniae Media
Friss Media
Hovind-Hougen, K., Friss, N. F., Research in Veterinary Science, 1991, 51, pp 155–163, “Morphological & Ultrastructural Studies of M flocculare and M hyopneumoniae in vitro”.
250 ml Hanks BSS
140 ml Water
1.5 gm Brain Heart infusion
1.6 gm PPLO Broth w/o CV
Autoclave at 120° C. for 20 minutes
18 ml Yeast Extract (100 g YSC-2 Sigma in 750 ml)
3.7 ml 0.2% DNA in 0.1% Na2CL3 
5.14 ml 1%-NAD
0.6 ml 1% Phenol red

    Adjust to pH 7.3 to 7.4
    Filter through 0.45 um, 0.2 um membrane, store at 4° C.
    Add sterile Horse or Pig serum to 20%
    and Antibiotics prior to use

Etheridge Media



Etheridge, J. R., Cottew, G. S., Lloyd, L. C., Australian Veterinary Journal, 1979, August 55, pp 356–359, “Isolation of Mycoplasma hyopneumoniae from lesions in experimentally infected pigs”.














Materials
For 600 mls









Hanks BSS
18.9 ml



Martleys Digest broth
1.28 gm



Heart Infusion broth
1.65 gm



Lactalbumin hydrolysate
2.21 gm



Glucose
4.41 gm



Yeast Extract autolysate
8.82 ml



Pig Serum (filtered)
163 ml



1% NAD
6.17 ml



1% Phenol red
1.32 ml



0.2% DNA in 0.1% Na2CO3
4.41 ml










    Make up to 600 ml with MQ water (about 350–400 ml)
    Adjust pH to 7.4 and filter through: 3.0 um, 0.8 um, 0.45 um, 0.2 um.
    Store at 4° C.

Development of Immune Sows



Cull sows and naive gilt (unmated sow designated Dookie).
Challenged on numerous occasions, with culture grown M. hyopneumoniae and lung homogenate. Given intranasally and intratracheally. Period of challenge—from September, 1991 to 21st Jan., 1992.
Tiamulin antibiotic given 31st Jan., 1992 to 4th Feb., 1992. Rested for approximately 8 weeks.
Infectious Challenge
120 ml of frozen culture of M. hyopneumoniae strain Beaufort, spun down (12,000×g, 20 min.) and resuspended in 50 ml complete medium and cultured overnight at 37° C. The overnight culture was centrifuged (12,000×g, 20 min.) and the mycoplasma cells resuspended in 10 ml serum free mycoplasma culture medium. The 10 ml of concentrated mycoplasma was administered to anaesthetized immune sows via a catheter to ensure the inoculum was placed into the trachea.
Three of four days post-challenge, the sows were killed, and lymph nodes draining the lungs taken—these included the left and right tracheobronchial lymph nodes, and the lymph nodes located at the bifurcation of the trachea.
Antibody probes were prepared from pig lymph nodes and utilized to detect putative protection antigens as described in Australian Patent Application 49035/90 referred to above. Separate cell cultures were obtained from individual lymph nodes. Culture supernatants were harvested after 5 days of culture.
Antigen Preparation
Mycoplasma hyopneumoniae strain Beaufort was cultured in Etheridge media until the pH had dropped to between 6.8 and 7.0. Cells of M. hyopneumoniae were harvested from culture by centrifugation at 12,000×g for 20 min., washed 4 times with either sterile PBS or 0.25 M NaCl and then the pelleted cells extracted with one of the following.
(i) Sodium Dodecyl Sulphate (SDS)
The cell pellet was resuspended in 0.2% SDS and extracted for 2 hours at 37° C. Insoluble material was pelleted from the extract at 12,000×g for 10 min. and the soluble extract run on SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
(ii) Triton X-114
The method of Bordier (J. Bio. Chem. 1981, 256:1604–1606) was used to selectively extract membrane proteins using the non-ionic detergent Triton X-114.
The cell pellet was resuspended in cold PBS to 2 mg/ml protein and a cold pre-condensed solution of TX-114 added to give a final concentration of 1% (v/v) TX-114. Extraction was achieved by incubation overnight at 4° C. with gentle mixing. Insoluble material was removed by centrifugation at 12,000×g for 20 min. at 4° C. The Triton X-114 soluble membrane proteins were then obtained by achieving a phase separation at 37° C.
Proteins soluble in TX-114 phase were precipitated with 80% ethanol in the presence of carrier dextran (80,000 molecular weight) at −70° C. overnight. The proteins were collected by centrifugation at 12,000×g for 30 min. and dissolved to 500 ug/ml in 4 M urea.
Identification of Antigens
Six antigens were identified utilizing the above-mentioned technique. The identified antigens were those that were consistently identified by the antibody probes from the immune cultures and the Dookie gilt. The results are summarized in Table 1.








TABLE 1





Molecular Weight (kD)
Characteristics







110–114
SDS Extracted


90–94
SDS Extracted


72–75
Triton X-114 Extracted


 60–64**
SDS Extracted. Partitions to aqueous



phase of Triton X-114 extract.


52–54
Triton X-114 Extracted


46–48
Triton X-114 Extracted





**Two antigens of approximate molecular weight 62 kD were identified.

















Molecular



Weight (kD)
Amino Acid Sequence

















46–48
48 K N-Terminal: AGXGQTESGSTSDSKPQAETLKHKV
(SEQ ID NO:3):



48 K CNBR F 1: TIYKPDKVLGKVAVEVLRVLIAKKNKASR
(SEQ ID NO:4):



48 K CNBR F 2: AEQAITKLKLEGFDTQ
(SEQ ID NO:5):



48 K CNBR F 3: KNSQNKIIDLSPEG
(SEQ ID NO:6):


52–54
52 K Terminal: AGXWAKETTKEEKS
(SEQ ID NO:7):



52 K CNBR F 1: AWVTADGTVN
(SEQ ID NO:8):



52 K CNBR F 2: AIVTADGTVNDNKPNQWVRKY
(SEQ ID NO:9):


60–64
52 K N-Terminal: MKLAKLLKGFX (N/L)(M/V) IK
(SEQ ID NO:10):


60–64
52 K N-Terminal: ADP(F/I)(R/E)Y(V/A)PQG(Q/A)X(M/N)VG
(SEQ ID NO:11):


72–75
74 K N-Terminal: AGXLQKNSLLEEVWYLAL
(SEQ ID NO:12):



74 K CNBR F 1: AKNFDFAPSIQGYKKIAHEL
(SEQ ID NO:13):



74 K CNBR F 2: NLKPEQILQLLG
(SEQ ID NO:14):



74 K CNBR F 3: LLKAEXNKXIEEINTXLDN
(SEQ ID NO:15):





CNBR - Cyanogen Bromide fragment.


X denotes an undetermined amino acid.


(A/B) - residue may be A or B






PCR of 48 kDa Gene

Polymerase Chain Reaction (PCR) oligonucleotide primers were designed from the amino acid sequences obtained from the N-terminal and internal cyanogen bromide (CNBr) derived peptides. Inosine (I) was substituted at positions of high redundancy. The following primers were used in a standard PCR assay, run on a Bartelt Gene Machine Robotic thermal cycling instrument.








Oligo 48 K CNBr F 1:
ACIAACGACGAGAAGCCICAGGC



     T  T  A  A     A


 


Oligo 48 K CNBr F 2:
TTIAGCTTIGTGATIGCCTGCTC



    AT    A     T  T



          T


 


Oligo 48 K CNBr F 3:
AGGTCGATGATCTTCCAICC



 AA  A  A  T  T



     T  T






The resulting PCR products were visualised on a 1.5% agarose gel, excised, and purified using Prep-a-Gene (BioRad). They were cloned by standard techniques into a dideoxy tailed T-vector (Holton and Graham, Nucleic Acids Research 19: 1156, 1991) and the nucleic acid sequence determined. The PCR product, obtained from the reaction using primers F1 and F2 shown above, was of approximately 810 base pairs and was shown by sequencing to code for the previously determined amino acid sequence of the purified native 46–48 kDa protein.

Genomic Clone Isolation of 48 k Gene

The entire 48 K gene has been isolated and sequenced. The gene was obtained from an M. hyopneumoniae genomic library made by digesting genomic DNA with the restriction enzyme CLA I and ligating the fragments into the vector pBluescript (Stratagene). The ligated product was then electroporated into Escherichia coli strain SURE (Stratagene) and the cells plated on Luria Broth agar plates containing 100 mg/ml Ampicillin (LB-Amp). The library was screened by DNA hybridisation with a polymerase chain reaction (PCR) product specific for the 48 kDa protein. Positive clones were grown in LB-Amp, the cells harvested and the DNA isolated and partially sequenced for confirmation.
The positive clone pC1-2 was entirely sequenced and the protein sequence deduced. This was compared to the protein sequence obtained from the N terminus and Cyanogen Bromide fragments of the 48 kDa protein to show that the gene encoded the desired protein.
Adjuvant Selection
Young piglets, 5–7 weeks of age, were immunized with identified antigen(s). The antigens include Triton X-114 extract and identified proteins of 46–48, 52–53, 60–64, 70–75, 90–94 and 110–114 kD, either singly or in combination. An immunizing dose of antigen, containing between 5–100 μg protein, was given by intramuscular injection in combination with an adjuvant. An adjuvant is selected from

    
    
        (i) Seppic Montanide ISA-50
        (ii) Quill A and other derivatives of saponin,
        (iii) oil in water emulsion employing a mineral oil such as Bayol F/Arlacel A,
        (iv) oil in water emulsion employing a vegetable oil such as corn oil, safflower oil or other with lecithin as emulsifier,
        (v) aluminium hydroxide gel, and
        (vi) nonionic block polymer such as Pluronic F-127 produced by BASF (U.S.A.).
    
    


Immunizing doses were given at 2–4 week intervals, the number of doses being dependent on the adjuvant and amount of antigen, but preferably 2 to 3 doses are given.
Adjuvants were treated on the basis of being able to induce antibody titers, as measured by ELISA, and by assessment of induced cell-mediated immunity as tested by Delayed-Type Hypersensitivity (DTH) reaction.
The results clearly show that mineral-oil type adjuvants are consistently superior at inducing antibody titers and DTH responses (Table 2). In particular an adjuvant marketed under trade designation Montanide ISA-50 and available from Seppic, Paris, France has been found to be suitable.











TABLE 2






Animal
DTH 24 Hour
DTH 48 Hour
Antibody Levels


GROUP
Number
Response
Response
(450 nm)



















CON-
19
0
0
0.061


TROL
11
0
0
0.010


(Un-
1
—
—
0.005


vaccin-
15
0
0
0.038


nated)
7
0
0
0.005


QUIL A
18
+
0
0.753



25
+
0
0.788



17
0
0
0.638



168
—
±
0.642


VEG.
169
+++
0
0.316


OIL
22
0
0
0.621



4
+
0
0.666



5
+
—
0.239



13
+++
++
0.457


MIN.
14
+++
++
1.085


OIL
5
+++
++
1.024



23
+++
+
0.864



15
+++
0
0.975



21
+
—
0.954





TABLE 2. Antibody levels and DTH responses in pigs measured 2 weeks after the third injection of antigen from M. hyopneumoniae. (— = no response; ± = faint reddening; + = faint reddening and swelling: ++ = reddining: +++ = swelling with or without reddening).






Protection Pen Trial

Groups of 9 young piglets, 6 weeks of age, were immunized with purified and semi-purified antigens as shown in Table 3 below. The antigens were purified on reversed-phase HPLC using a formic acid solvent system with an acetonitrile gradient.
Antigens were resolubilized in 4 Molar urea before incorporation in mineral oil adjuvant.
The immunization schedule is as shown in Table 2.







TABLE 3





Protocol for Pen Trial of Antigens of Mycoplasma Hyopneumoniae


















VACCINATIONS & BLEEDS




Treatment
Day Number







1st Vaccination
 0



2nd Vaccination
14



3rd Vaccination
50



Infectious Challenge
64



Slaughter
91












ANTIGEN DOSES





Partly Purified
1st & 2nd Vaccns. 50 μg COMPLEX ANTIGEN/



DOSE


52 kD
3rd Vaccn. - 220 μg PARTIALLY PURIFIED



ANTIGEN/DOSE


(Purified)74 + 52 kD
1st Vaccn. 20 μg total protein/DOSE



2nd Vaccn. 13 μg total protein/DOSE



3rd Vaccn. 17 μg total protein/DOSE


(Purified) 48 KD
1st Vaccn. 20 μg/DOSE



2nd Vaccn. 18 μg/DOSE



3rd Vaccn. 27 μg/DOSE









All Protein Estimations Done by “BCA” Protein Assay (Pierce, Ill., U.S.A.

Protection from infection with Mycoplasma hyopneumoniae was assessed by infectious challenge 2 weeks after the final immunization. Infectious challenge was achieved by intranasal administration of 10 ml of a 10% (w/v) lung homogenate, prepared from infected lung, and by housing test piglets with previously infected piglets. Four weeks after infectious challenge, the animals were killed and the extent and degree of lung lesions assessed (Table 4).








TABLE 4







Pen Trial of Antigens of Mycoplasma Hyponeumoniae











No. Pneumonia
Medium Lung
% Reduction


Group No.
Free (%)
Lesion Score
(from Median)













Controls
1 (11)
13
0%


52 kD
0 (0) 
5
61%


74 ÷ 52
3 (33)
6.75
48%


kD


48 kD
2 (22)
6.25
52%









REFERENCE


    Warren H. S. and Chedid, L. A., Future Prospects for Vaccine Adjuvants CRC Critical Reviews in Immunology 8: 83–108, 1988.


Finally, it is to be understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as outlined herein.

## Claims

1. An isolated antigen prepared by a method comprising:
(a) providing a sample of a Mycoplasma, 
(b) providing an antibody probe including at least one antibody against the Mycoplasma, said at least one antibody being produced by a method comprising
(i) providing a biological sample taken after a mammal has been challenged with the Mycoplasma or an extract comprising the Mycoplasma at an infection or lesion site, said biological sample being taken from the infection or lesion site or an area close to the infection or lesion site, wherein the biological sample is taken from the mammal within about 2 to 5 days after the mammal has been challenged with the Mycoplasma or extract;
(ii) isolating antibody producing cells from the biological sample;
(iii) culturing the isolated cells in vitro in suitable culture medium; and
(iv) harvesting the at least one antibody from said cultured cells;

(c) probing the Mycoplasma sample with the antibody probe to detect at least one antigen; and
(d) isolating the at least one antigen detected.

2. An isolated antigen comprising a molecular structure that is identifiable with an antibody probe produced by harvesting an antibody from antibody producing cells of a mammal that are at or close to an infection or lesion site within 2 to 5 days after said mammal is challenged by infection with Mycoplasma hyopneumoniae at said infection or lesion site, said molecular structure being a native Mycoplasma hyopneumoniae antigen having an approximate molecular weight in kilodaltons (kD) of between 110–114, 90–94, 72–75, 52–54 or 46–48, or being a mutant, derivative or fragment of the native antigen that stimulates production of the antibody in the antibody producing cells, wherein if the molecular structure is the native antigen having the molecular weight between 72–75 kD, the molecular structure contains an N-terminal amino acid sequence comprising SEQ ID NO:12, and wherein if the molecular structure has a molecular weight between 46–48 kD, the molecular structure has an N-terminal amino acid sequence comprising SEQ ID NO:3.

3. An isolated antigen according to claim 2, wherein the molecular structure comprises the N-terminal amino acid sequence comprising SEQ ID NO:12.

4. An isolated antigen according to claim 3, comprising at least one internal amino acid sequence selected from the group consisting of SEQ ID NO13; SEQ ID NO:14 and SEQ ID NO:15.

5. An isolated antigen according to claim 2, wherein the molecular structure has a molecular weight between 60–64 kD and has an N-terminal amino acid sequence comprising SEQ ID NO:10 or SEQ ID NO:11.

6. An isolated antigen according to claim 2, wherein the molecular structure has a molecular weight between 52–54 kD and has an N-terminal amino acid sequence comprising SEQ ID NO:7.

7. An isolated antigen according to claim 6, comprising at least one internal amino acid sequence selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:9.

8. An isolated antigen according to claim 2, wherein the molecular structure has a molecular weight between 46–48 DK and has an N-terminal amino acid sequence comprising SEQ ID NO:3.

9. An isolated antigen according to claim 8, comprising at least one internal amino acid sequence from the group consisting of SEQ ID NO:4; SEQ ID NO:5 and SEQ ID NO:6.

10. A method for preparing a synthetic antigenic polypeptide against Mycoplasma, which method comprises
(a) providing a cDNA library or genomic library derived from a sample of the Mycoplasma; 
(b) providing an antibody probe produced by
(i) providing a biological sample taken after a mammal has been challenged with the Mycoplasma or an extract comprising the Mycoplasma at an infection or lesion site, said biological sample being taken from the infection or lesion site or an area close to the infection or lesion site, wherein the biological sample is taken from the mammal within about 2 to 5 days after the mammal has been challenged with the Mycoplasma or extract;
(ii) isolating antibody producing cells from the biological sample;
(iii) culturing the isolated cells in vitro in a suitable culture medium; and
(iv) harvesting at least one antibody from said isolated cells;

(c) generating synthetic polypeptides from the cDNA library or genomic library;
(d) probing the synthetic polypeptides with the antibody probe to detect the synthetic antigenic polypeptide; and
(e) isolating the synthetic antigenic polypeptide detected thereby.

11. A method according to claim 10, wherein the at least one antibody is raised against an antigen from Mycoplasma hyopneumoniae or a related organism, said antigen being selected from the group of native Mycoplasma antigens having approximate molecular weights of 110–114, 90–94, 72–75, 52–54 and 46–48 kilodaltons (kD) or being a mutant, derivative or fragment of a native Mycoplasma antigen that stimulates production of the at least one antibody in said mammal.

12. A synthetic antigen produced by the method of claim 10.

13. A vaccine or veterinary composition comprising a prophylactically effective amount of at least one antigen according to claim 2.

14. A vaccine or veterinary composition comprising prophylactically effective amounts of a plurality of antigens according to claim 2.

15. A diagnostic kit including an antigen according to claim 2.

16. A method for preventing or treating Mycoplasma infection, which method comprises administering to a mammal a prophylactically or therapeutically effective amount of at least one antigen according to claim 2.

17. An amino acid sequence encoded by a SEQ ID NO:1.

18. An amino acid sequence consisting of SEQ ID NO:2.