ANTIGEN COMPOSITION AGAINST MYCOPLASMA

Description

[0001] 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.

[0002]

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.

[0003] Whilst several experimental vaccines have been produced, these have resulted in less than optimal results, and utilising 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.

[0004] European Patent Application 359 919 to ML Technology Ventures L.P. describes a series of antigens, 36 kD, 41 kD 74.6 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.

[0005] However, there remains a need in the art for an effective vaccine against M. hyopneumoniae which would confer protection against colonisation and clinical disease following M. hyopneumoniae challenge and also significantly reduce the morbidity and mortality from secondary infections.

[0006] 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.

[0007] Accordingly, in a first aspect of the present invention there is provided a putative protective antigen against a Mycoplasma, preferably Mycoplasma hyopneumoniae prepared by a method including

[0008] providing

[0009] a sample of a Mycoplasma;

[0010] an antibody probe including at least one antibody against a Mycoplasma produced by a method including:

[0011] 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;

[0012] isolating cells from the biological sample;

[0013] culturing cells in vitro in a suitable culture medium; and

[0014] harvesting antibodies produced from said cells;

[0015] probing the Mycoplasma sample with the antibody probe to detect at least one antigen; and

[0016] isolating the antigen detected.

[0017] The protective antigens may also function as diagnostic antigens as discussed below.

[0018] Accordingly, in a preferred aspect of the present invention there is provided a putative protective antigen against Mycoplasma hyopneumoniae, or related infections, selected from the group of 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 putative protective antigen may be a surface protein. The putative protective antigen may be a surface lipoprotein or membrane protein.

[0019] Preferably the protective antigens are selected from the group of antigens having approximate molecular weights of 110-114, 90-94, 74, 62, 52 and 48 kD.

[0020] Preferably, the 72-75 kD antigen includes the following N-terminal amino acid sequence:

[0021] AGXLQKNSLLEEVWYLAL

[0022] and, optionally, one or more of the following internal amino acid sequences:

[0023] AKNFDFAPSIQGYKKIAHEL

[0024] NLKPEQILQLLG

[0025] LLKAEXNKXIEEINTXLDN

[0026] Preferably, the 60-64 kD antigen includes one of the following N-terminal amino acid sequences:

[0027] MKLAKLLKGFX(N/L)(M/V)IK

[0028] ADP(F/I)(R/E)Y(V/A)PQG(Q/A)X(M/N)VG

[0029] Preferably, the 52-54 kD antigen includes the following N-terminal amino acid sequence:

[0030] AGXWAKETTKEEKS

[0031] and, optionally, one or more of the following internal amino acid sequences:

[0032] AWVTADGTVN

[0033] AIVTADGTVNDNKPNQWVRKY.

[0034] Preferably, the 46-48 kD antigen includes the following N-terminal amino acid sequence:

[0035] AGXGQTESGSTSDSKPQAETLKHKV

[0036] and, optionally, one or more of the following internal amino acid sequences:

[0037] TIYKPDKVLGKVAVEVLRVLIAKKNKASR

[0038] AEQAITKLKLEGFDTQ

[0039] KNSQNKIIDLSPEG

[0040] The 46-48 kD antigen may be encoded by a nucleic acid fragment:

110 20 30 40 501234567890 1234567890 1234567890 1234567890 1234567890ATGAAAAAAA TGACACTATA CCAGAGGAAA GAGCAGTATA TAAAATAATT50AAAATTACAT TTTCTTCAAT TGCGCCAGAA TTTTTAAGAA TTAGTACATT100AAAAAGTAGA ACAAAAGTTA TTAATGTAAA CATTAGCGCA ATCCTTAAGA150AAAAATTAAA AGTTTTATCT ATTTTTTTTA ATCGAAATCC AACCAGGCAT200AAATCTTTGT CAGTATTTAT CAAGTCGGTA TTTTTTCATT ATTTCTACTA250AAATATTATT TGAATTTGCA TTTTCCATAA TCTAAAATTT TACATTTTTT200TATAACAATT TTTAAAAATT ACTCTTTAAT TTATAGTATT TTTTTATTTT350TTAGTCTAAA TTATAAAATT ATCTTGAATT TTATTTGAAT TTTTATAATT400TAGTACTAAA AAATACAAAT ATTTTTTACT ATTCTAAGAA AAATTCATTT450TTAAAAAAAA ATTGATTTTT ATAGTATAAT TTGTTTGTAT AATTGAATTA500ACTTGATTTG AAAGGGAACA AAATGAAAAA AATGCTTAGA AAAAAATTCT550TGTATTCATC AGCTATTTAT GCAACTTCGC TTGCATCAAT TATTGCATTT500GTTGCAGCAG GTTGTGGACA GACAGAATCA GGTTCAACTT CTGATTCTAA550ACCACAAGCG GAGACGCTAA AACATAAAGT AAGTAATGAT TCTATTCGAA700TAGCACTAAC CGATCCGGAT AATCCTCGAT GAATTAGTGC CCAAAAAGAT750ATTATTTCTT ATGTTGATGA AACAGAGGCA ACAACTTCAA CAATTACAAA800AAACCAGGAT GCACAAAATA AATGACTCAC TCAGCAAGCT AATTTAAGCC850CAGCGCCAAA AGGATTTATT ATTGCCCCTG AAAATGGAAC TGGAGTTGGA900ACTGCTGTTA ATACAATTGC TGATAAACGA ATTCCGATTG TTGCCTATGA950TCGACTAATT ACTGGATCTG ATAAATATGA TTGGTATGTT TCTTTTGATA1000ATGAAAAAGT TGGTGAATTA CAAGGTCTTT CACTTGCTGC GGATCTATTA1050GGAAAAGAAG ATGGTGCTTT TGATTCAATT GATCAAATGA ATGAATATCT1100AAAATCACAT ATGCCCCAAG AGACAATTTC TTTTTATACA ATCGCGGGTT1150CCCAAGATGA TAATAATTCC CAATATTTTT ATAATGGTGC AATGAAAGTA1200CTTAAAGAAT TAATGAAAAA TTCGCAAAAT AAAATAATTG ATTAATCTCC1250TGAAGGCGAA AATGCTGTTT ATGTCCCAGG ATGAAATTAT GGAACTGCCG1300GTCAAAGAAT CCAATCTTTT CTAACAATTA ACAAAGATCC AGCAGGTGGT1350AATAAAATCA AAGCTGTTGG TTCAAACCAG GCTTCTATTT TCAAAGGATT1400TCTTGCCCCA AATGATGGAA TGGCCGAACA AGCAATCACC AAATTAAAAC1450TTGAAGGGTT TGATACCCAA AAAATCTTTC TAACTCGTCA AGATTATAAT1500GATAAAGCCA AAACTTTTAT CAAAGACGGC GATCAAAATA TGACAATTTA1550TAAACCTGAT AAAGTTTTAG GAAAAGTTGC TGTTGAAGTT CTTCGGGTTT1600TAATTGCAAA GAAAAATAAA GCATCTAGAT CAGAAGTCGA AAACGAACTA1650AAAGCAAAAC TACCAAATAT TTCATTTAAA TATGATAATC AAACATATAA1700AGTACAAGGT AAAAATATTA ATACAATTTT AGTAAGTCCA GTAATTGTTA1750CAAAAGCTAA TGTTGATAAT CCTGATGCCT AA1732

[0041] 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:

210 20 30 40 501234567890 1234567890 1234567890 1234567890 1234567890ATGAAAAAAA TGCCACTATA CCAGAGGAAA GAGCAATATA TAAAATAATT50AAAATTACAT TTTCTTCATT TGCGCCAGAA TTTTTAAGAA TTAGTACATT100AAAAAGTAGA ACAAAAGTTA TTAATGTAAA CATTAGCACA ATCCTTAAGA150AAAAATTAAA AGTTTTATCT ATTTTTTTTA TCGAAAATCC AACCAGGCAT200AAATCTTTGT CAGTATTTAT CAAGTCGGTA TTTTTTCATT ATTTCTACTA250AAATATTATT TGAATTTGCA TTTTCCATAA TCTAAAAATT TACATTTTTT300TATAACAATT TTTAAAAATT ACTCTTTAAT TTATAGTATT TTTTTATTTT350TTAGTCTAAA TTATAAAATT ATCTTGAATT TTATTTGAAT TTTTATAATT400TAGTACTAAA AAATACAAAT ATTTTTTCCT ATTCTAAGAA AAATTCAATT450TTTAAAAAAA ATTGATTTTT ATAGTATAAT TTGTTTGTAT AATTGAATTA500ACTTGATTTG AAAGGGAACA AATAAAAAAA AATGCTTAGA AAAAAATTCT550TGTATTCATC AGCTATTTAT GCAACTTCGC TTGCATCAAT TATTGCATTT500GTTGCAGCAG GTTGTGGACA GACAAAATCA GGTTCAACTT CTGATTCTAA550ACCACAAGCC GAGACGCTAA AACATAAAGT AAGTAATGAT TCTATTCGAA700TAGCACTAAC CGATCCAGAT AATCCTCGAT GAATTAGTGC CCAAAAAGAT750ATTATTTCTT ATGTTAATAA AACAAAGGCA GCAACTTCAA CAATTACAAA800AAACCAGAAT GCACAAAATA ACTGACTCAC TCAGCAAGCT AATTTAAGCC850CAGCGCCAAA AGGATTTATT ATTGCCCCTG AAAATGGAAG TGGAGTTGGA900ACTCCTGTTA ATACAATTGC TGATAAAGGA ATTCCGATTG TTGCCTATGA950TCGACTAATT ACTGGATCTG ATAAATATAA TTGGTATGTT TCTTTTGATA1000ATAGAAAAGT TGGTGAATTA CAAAGTCTTT CACTTGCTGC GGGTCTATTA1050GGAAAAGAAG ATGGTGCTTT TGATTCAATT GATCAAATGA ATGAATATCT1100AAAATCACAT ATGCCCCAAG AGACAATTTC TTTTTATACA ATCGCGGGTT1150CCCAAGATGA TAATAATTCC CAATATTTTT ATAATGGTGC AATGAAAGTA1200CTTAAAGAAT TAATGAAAAA TTCGCAAAAT AAAATAATTG ATTTATCTCC1250TGAAGGCGAA AATGCTGTTT ATGTCCCAGG ATGAAATTAT GGAACTGCCG1300GTCAAAGAAT CCAATCTTTT CTAACAATTA ACAAAGATCC AGCAGGTGGT1350AATAAAATCA AAGCTGTTGG TTCAAAACCA GCTTCTATTT TCAAAGGATT1400TCTTGCCCCA AATGATCGAA TGGCCAAACA AGCAATCACC AAATTAAAAC1450TTGAAGGGTT TGATACCCAA AAAATCTTTG TAACTCGTCA ACATTATAAT1500GATAAAGCCA AAACTTTTAT CAAAGACGGC GATCAAAATA TGACAATTTA1550TAAACCTGAT AAAGTTTTAG GAAAAGTTGC TGTTGAAGTT CTTCGGGTAT1600TAATTGCAAA GAAAAATAAA GCATCTAGAT CAGAAGTCGA TAACGAACTA1650AAAGCAAAAC TACCAAATAT TTCATTTAAA TATGATAATC AAACATATAA1700AGTACAAGGT AAAAATATTA ATACAATTTT AGTAAGTCAA GTAATTGTCA1750CAAAAGCTAA TGTTGATAAT CCTGATGCCT AA1752

[0042] 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).

[0043] 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.

[0044] The present invention further provides a vaccine composition including a prophylactically effective amount of at least one putative protective antigen against a Mycoplasma as herein described. Preferably the veterinary composition includes two or more putative protective antigens as herein described.

[0045] Accordingly in a preferred aspect the present invention provides a vaccine composition including two or more putative protective antigens selected from antigens having approximate molecular weights of 110-114, 90-94, 72-75, 60-64, 52-54 and 46-48 kilodaltons.

[0046] The vaccine composition may include any combination of two or more putative 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.

[0047] 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.

[0048] 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.

[0049] 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 phsyiological concentrations, and solid carriers, such as talc or sucrose.

[0050] 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.

[0051] More preferably the adjuvant is of the mineral-oil type as these have been found to be consistently superior at inducing antibody titres 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.

[0052] When used for administering via the bronchial tubes, the vaccine is suitably present in the form of an aerosol.

[0053] 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.

[0054] The putative protective antigens according to the present invention may be isolated and identified utilising the general methods described in Australian patent application 49035/90, the entire disclosure of which is incorporated herein by reference.

[0055] Accordingly, in a further aspect, the present invention provides a method for producing at least one antibody against a Mycoplasma. This method includes

[0056] 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;

[0057] isolating cells from the biological sample;

[0058] culturing cells in vitro in a suitable culture medium; and

[0059] harvesting antibodies produced from said cells.

[0060] The Mycoplasma may be Mycoplasma hyopneumoniae.

[0061] The animal may be a mammal including humans. The mammal may be a domestic animal such as a pig, sheep or cattle.

[0062] 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.

[0063] 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.

[0064] In contrast, the probes described in the present invention are highly enriched in Mycoplasma-specific antibodies of particular importance to protective immunity.

[0065] 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.

[0066] The cells isolated from the biological sample may include B cells.

[0067] 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.

[0068] 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.

[0069] The method of producing an antibody may include a further step of activating the cells isolated to proliferate and secrete and/or release antibodies.

[0070] 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.

[0071] The mitogens may be selected from the group including 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 selected from the group including 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.

[0072] 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.

[0073] 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

[0074] providing

[0075] a sample of a Mycoplasma; and

[0076] an antibody probe including at least one antibody against a Mycoplasma;

[0077] probing the Mycoplasma sample with the antibody probe to detect at least one antigen; and

[0078] isolating the antigen detected.

[0079] 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).

[0080] Alternatively the proteins may be selected utilising 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).

[0081] The separate proteins may then be transferred to nitrocellulose, nylon or other sheets.

[0082] 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).

[0083] 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.

[0084] 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.

[0085] Accordingly, in a preferred aspect there is provided a method for purifying antigen. This method includes

[0086] providing

[0087] a crude antigen mixture; and

[0088] an antibody against a Mycoplasma immobilized on a suitable support;

[0089] subjecting the crude antigen mixture to affinity chromatography utilizing the immobilized antibody; and

[0090] isolating the purified antigen so formed.

[0091] The antibody is produced by the method described above.

[0092] 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 4S (Pharmacia), Affi-gel (Bio-RAD), or other affinity chromatography supports able to bind proteins.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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

[0097] providing

[0098] a cDNA library or genomic library derived from a sample of Mycoplasma; and

[0099] an antibody probe as described above;

[0100] generating synthetic polypeptides from the cDNA library or genomic library;

[0101] probing the synthetic polypeptides with the antibody probe; and

[0102] isolating the synthetic antigenic polypeptide detected thereby.

[0103] 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

[0104] (i) synthetic oligonucleotide probes based on the amino acid sequence of the antigen identified and purified as described above;

[0105] (ii) antibodies obtained from the culture medium produced as described above;

[0106] (iii) monoclonal or polyclonal antibodies produced against the antigens identified and purified as described above;

[0107] (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).

[0108] The synthetic antigenic polypeptide produced in accordance with the invention may be a fusion protein containing the synthetic antigenic peptide and another protein.

[0109] 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.

[0110] 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.

[0111] Preferably the clone is pC1-2.

[0112] 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.

[0113] In the figures:

[0114]
FIG. 1: SDS-Polyacrylamide gel (12.5%) profiles of SDS extracts of species of mycoplasma—Coomassie R250 stained.

3Lane 1Pre-stained Molecular Weight StandardsLane 2M. gellisepticumLane 3M. synoviae.Lane 4M. hyopneumoniae.Lane 5M. hyorhinis.Lane 6M. flocculare.

[0115]
FIG. 2: SDS-Polyacrylamide gel (12.5%) profiles of extracts of strains of M. hyopneumoniae—Coomassie R250 stained gel

4Lane 1Pre-stained Molecular Weight Standards.Lane 2Triton X-114 extract of M. hyopneumoniae - strain Beaufort.Lane 3As for Lane 2.Lane 4SDS extract of M. hyopneumoniae strain Beaufort.Lane 5SDS extract of M. hyopneumoniae strain 10110.

[0116]
FIG. 3: Western blots of Triton X-114 extracted antigens from M. hyopneumoniae strain Beaufort, probed with serum and supernatant antibody probes.

5Lane 1No antibody controlLane 2Dookie pig serum control 1/200.Lane 3Pig 105 supernatant.Lane 4Pig 1 supernatant.Lane 5Dookie pig supernatant.

[0117]
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%).

6Lane 1a) Pig 453 supernatant.b) Pig 453 serum 1/100.Lane 2a) Pig 105 supernatant.b) Pig 105 serum 1/100.Lane 3a) Pig 1 supernatant.b) Pig 1 serum1/100.Lane 4a) Pig 15 supernatant.b) Pig 15 serum 1/100.Lane 5a) Dookie supernatant.b) Dookie serum 1/100.Lane 6No antibody control.

[0118]
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%).

7Lane 1a) Pig 453 supernatant.b) Pig 453 serum 1/100.Lane 2a) Pig 105 supernatant.b) Pig 105 serum 1/100.Lane 3a) Pig 1 supernatant.b) Pig 1 serum 1/100.Lane 4a) Pig 15 supernatant.b) Pig 15 serum 1/100.Lane 5a) Dookie supernatant.b) Dookie serum 1/100.Lane 6No antibody control.

[0119]
FIG. 6: The entire 48 k gene sequence.

[0120]
FIG. 7: tHE 48 kDa protein sequence of the 48 k gene sequence.

EXAMPLE 1

Mycoplasma hyopneumoniae
Media

[0121] Friss Media

[0122] Hovind-Hougen, K., Friss, N. F., Research in Veterinary Science, 1991, 51, pp 156-163, “Morphological & Ultrastructural Studies of M. flocculare and M. hyopneumoniae in vitro”.

[0123] 250 ml Hanks BSS

[0124] 140 ml Water

[0125] 1.5 gm Brain Heart infusion

[0126] 1.6 gm PPLO Broth w/o CV

[0127] Autoclave at 120° C. for 20 minutes

[0128] 18 ml Yeast Extract (100 g YSC-2 Sigma in 750 ml)

[0129] 3.7 ml 0.2% DNA in 0.1% Na2CL3

[0130] 5.14 ml 1%-NAD

[0131] 0.6 ml 1% Phenol red

[0132] Adjust to pH 7.3 to 7.4

[0133] Filter through 0.45 um, 0.2 um membrane, store at 4° C.

[0134] Add sterile Horse or Pig serum to 20%

[0135] and Antibiotics prior to use

[0136] Etheridge Media

[0137] Etheridge, J. R., Cottew, G. S., Lloyd, L. C., Australian Veterinary Journal, August 1979, 56, pp 356-359, “Isolation of Mycoplasma hyopneumoniae from lesions in experimentally infected pigs”.

8MaterialsFor 600 mlsHanks BSS18.9 mlMartleys Digest broth1.28 gmHeart Infusion broth1.65 gmLactalbumin hydrolysate2.21 gmGlucose4.41 gmYeast Extract autolysate8.82 mlPig Serum (filtered)163 ml1% NAD6.17 ml1% Phenol red1.32 ml0.2% DNA in 0.1% Na2CO34.41 ml

[0138] Make up to 600 ml with MQ water (about 350-400 ml)

[0139] Adjust pH to 7.4 and filter through: 3.0 um, 0.8 um 0.45 um, 0.2 um.

[0140] Store at 4° C.

[0141] Development of Immune Sows

[0142] Cull sows and naive gilt (unmated sow designated Dookie).

[0143] Challenged on numerous occasions, with culture grown M. hyopneumoniae and lung homogenate. Given intranasally and intratracheally. Period of challenge—from September 1991 to Jan. 21, 1992.

[0144] Tiamulin antibiotic given Jan. 31, 1992 to Feb. 4, 1992. Rested for approximately 8 weeks.

[0145] Infectious Challenge

[0146] 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 anaesthetised immune sows via a catheter to ensure the inoculum was placed into the trachea.

[0147] 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.

[0148] Antibody probes were prepared from pig lymph nodes and utilised 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.

[0149] Antigen Preparation

[0150]

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.

[0151] (i) Sodium Dodecyl Sulphate (SDS)

[0152] 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).

[0153] (ii) Triton X-114

[0154] 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.

[0155] 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.

[0156] 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.

[0157] Identification of Antigens

[0158] Six antigens were identified utilising 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 summarised in Table 1.

9TABLE 1Molecular Weight (kD)Characteristics110-114SDS Extracted90-94SDS Extracted72-75Triton X-114 Extracted 60-64**SDS Extracted. Partitions to aqueousphase of Triton X-114 extract.52-54Triton X-114 Extracted46-48Triton X-114 Extracted**Two antigens of approximate molecular weight 62 kD were identified.

[0159]

10

Molecular

Weight (kD)
Amino Acid Sequence

46-48
48 K N-Terminal: AGXGQTESGSTSDSKPQAETLKHKV

48 K CNBR F 1: TIYKPDKVLGKVAVEVLRVLIAKKNKASR

48 K CNBR F 2: AEQAITKLKLEGFDTQ

48 K CNBR F 3: KNSQNKIIDLSPEG

52-54
52 K Terminal: AGXWAKETTKEEKS

52 K CNBR F 1: AWVTADGTVN

52 K CNBR F 2: AIVTADGTVNDNKPNQWVRKY

60-64
52 K N-Terminal: MKLAKLLKGFX (N/L)(M/V) IK

60-64
52 K N-Terminal: ADP(F/I)(R/E)Y(V/A)PQG(Q/A)X(M/N)VG

72-75
74 K N-Terminal: AGXLQKNSLLEEVWYLAL

74 K CNBR F 1: AKNFDFAPSIQGYKKIAHEL

74 K CNBR F 2: NLKPEQILQLLG

74 K CNBR F 3: LLKAEXNKXIEEINTXLDN

CNBR - Cyanogen Bromide fragment

X denotes an undetermined amino acid

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

[0160] PCR of 48 kDa Gene

[0161] 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.

11Oligo 48 K CNBr F A:ACIAACGACGAGAAGCCICAGGC T T A A AOligo 48 K CNBr F 2:TTIAGCTTIGTGATIGCCTGCTC AT A T T TOligo 48 K CNBr F 3:AGGTCGATGATCTTCCAICC AA A A T T T T

[0162] The resulting PCR products were visualised on a 1.5% agarose gel, excised, and purified using a 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.

[0163] Genomic Clone Isolation at 48 k Gene

[0164] The entire 48 k gene and 48 kDa protein (FIGS. 6 and 7) 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 1 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 μg/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.

[0165] 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 the that the gene encoded the desired protein.

[0166] Adjuvant Selection

[0167] Young piglets, 5-7 weeks of age, were immunised 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 immunising dose of antigen, containing between 6-100 μg protein, was given by intramuscular injection in combination with an adjuvant. An adjuvant is selected from

[0168] (i) Seppic Montanide ISA-50

[0169] (ii) Quill A and other derivatives of saponin,

[0170] (iii) oil in water emulsion employing a mineral oil such as Bayol F/Anacel A,

[0171] (iv) oil in water emulsion employing a vegetable oil such as corn oil safflower oil or other with lecithin as emulsifier,

[0172] (v) aluminium hydroxide gel, and

[0173] (vi) nonionic block polymer such as Pluronic F-127 produced by BASF (U.S.A.).

[0174] Immunising 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.

[0175] Adjuvants were treated on the basis of being able to induce antibody titres, as measured by ELISA, and by assessment of induced cell-mediated immunity as tested by Delayed-Type Hypersensitivity (DTH) reaction.

[0176] The results clearly show that mineral-oil type adjuvants are consistently superior at inducing antibody titres 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.

12TABLE 2AnimalDTH 24 HourDTH 48 HourAntibody LevelsGROUPNumberResponseResponse(450 nm)CON-19000.061TROL11000.010(Un-1——0.005vaccin-15000.038nated)7000.005QUIL A18+00.75325+00.78817000.638168——0.642VEG.169+++00.316OIL22000.6214+00.6665+—0.23913+++++0.457MIN.14+++++1.085OIL5+++++1.02423++++0.86415+++00.97521+—0.954TABLE 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).

[0177] Protection Pen Trial

[0178] Groups of 9 young piglets, 6 weeks of age, were immunised 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.

[0179] Antigens were resolubilised in 4 Molar urea before incorporation in mineral oil adjuvant.

[0180] The immunisation schedule is as shown in Table 2.

13TABLE 3Protocol for Pen Trial of Antigens of Mycoplasma HyopneumoniaeVACCINATIONS & BLEEDSTreatmentDay Number1st Vaccination 02nd Vaccination143rd Vaccination50Infectious Challenge64Slaughter91ANTIGEN DOSESPartly Purified1st & 2nd Vaccns. 50 μg COMPLEX ANTIGEN/DOSE52 kD3rd Vaccn. - 220 μg PARTIALLY PURIFIEDANTIGEN/DOSE(Purified)74 + 52 kD1st Vaccn. 20 μg total protein/DOSE2nd Vaccn. 13 μg total protein/DOSE3rd Vaccn. 17 μg total protein/DOSE(Purified) 48 KD1st Vaccn. 20 μg/DOSE2nd Vaccn. 18 μg/DOSE3rd Vaccn. 27 μg/DOSE

[0181] ALL PROTEIN ESTIMATIONS DONE BY “BCA” PROTEIN ASSAY (Pierce, Ill., U.S.A.

[0182] Protection from infection with Mycoplasma hyopneumoniae was assessed by infectious challenge 2 weeks after the final immunisation. 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).

14TABLE 4Pen Trial of Antigens of Mycoplasma HyponeumoniaeNo. PneumoniaMedium Lung% ReductionGroup No.Free (%)Lesion Score(from Median)Controls1 (11)130%52 kD0 (0) 561%74 ÷ 523 (33)6.7548%kD48 kD2 (22)6.2552%

[0183] Reference

[0184] Warren H. S. and Chedid, L. A., Future Prospects for Vaccine Adjuvants CRC Critical Reviews in Immunology 8: 83-108, 1988.

[0185] 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. A putative protective antigen against a Mycoplasma, 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.
2. A putative protective antigen according to claim 1 wherein the Mycoplasma is Mycoplasma hyopneumoniae.
3. A putative protective antigen against Mycoplasma hyopneumoniae, or related infections, selected from the group of antigens having approximate molecular weights of 110-114, 90-94, 72-75, 60-64, 52-54 and 46-48 kilodaltons (kD), as herein described, mutants, derivatives and fragments thereof.
4. A putative protective antigen according to claim 3 which is a surface protein.
5. A putative protective antigen according to claim 3 or 4 which is a surface lipo-protein or membrane protein.
6. A putative protective antigen according to any one of claims 3-5 having approximate molecular weight of 110-114, 90-94, 74, 62, 52 and 48 kD.
7. A putative protective antigen according to claim 3 wherein the antigen in the 72-75 kD region contains the following N-terminal amino acid sequence: AGXLQKNSLLEEVWYLAL
8. A putative protective antigen according to claim 7 further including one or more of the following N-terminal amino acid sequences: AKNFDFAPSIQGYKKIAHEL NLKPEQILQLLG LLKAEXNKXIEEINTXLDN
9. A putative protective antigen according to claim 3 wherein the antigen in the 60-64 kD region contains the following N-terminal amino acid sequence: MKLAKLLKGFX(N/L)(M/V)IK ADP(F/I)(R/E)Y(V/A)PQG(Q/A)X(M/N)VG
10. A putative protective antigen according to claim 3 wherein the antigen in the 52-54 kD region contains the following N-terminal amino acid sequence: AGXWAKETTKEEKS
11. A putative protective antigen according to claim 10 further including one or more of the following N-terminal amino sequences: AWVTADGTVN AIVTADGTVNDNKPNQWVRKY
12. A putative protective antigen according to claim 3 wherein the antigen in the 46-48 kD region contains the following N-terminal amino acid sequence: AGXGQTESGSTSDSKPQAETLKHKV
13. A putative protective antigen according to claim 12 further including one or more of the following internal amino acid sequences: TIYKPDKVLGKVAVEVLRVLIAKKNKASR AEQAITKLKLEGFDTQ KNSQNKIIDLSPEG
14. An isolated nucleic acid fragment encoding a putative protective antigen against Mycoplasma hyopneumoniae or related infections, said nucleic acid fragment including the following sequence, mutants, derivatives, recombinants and fragments thereof:
15. An isolated nucleic acid fragment according to claim 14 encoding putative protective antigen wherein the antigen is in the 46-48 kD region including the following nucleic acid sequence, mutants, derivatives, recombinants and fragments thereof:
16. A method for producing an antibody against a Mycoplasma 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 a 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.
17. A method according to claim 16 wherein the biological sample is taken at a predetermined time after the animal has been challenged with a Mycoplasma, preferably 2 to 7 days after challenge.
18. A method according to claim 16 wherein the culturing of cells in vitro further includes addition of helper factors to the culture, said helper factors selected from the group including 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.
19. A method according to any one of claims 16-18 further including a cell activation step including activating the cells isolated to proliferate and secrete and/or release antibodies said cell activation step including adding a cell activating agent to the culture medium, said cell activating agent selected from the group including mitogens as herein described and helper factors produced by leukocytes, or their synthetic equivalents or combinations thereof.
20. A method according to any one of claims 16-19 wherein the antibody is in the form of the supernatant harvested from the culture medium.
21. An antibody against a Mycoplasma prepared according to the method of any one of claims 16-20.
22. A method of identifying a putative protective antigen associated with a Mycoplasma, preferably Mycoplasma hyopneumoniae, said method including 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.
23. A method of purifying a putative protective antigen associated with a Mycoplasma, preferably Mycoplasma hyopneumoniae, said method including 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.
24. 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 including an antibody prepared according to claim 16;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.

Priority Claims (1)

Number	Date	Country	Kind
PN 1789	Mar 1995	AU

PCT Information

Filing Document	Filing Date	Country	Kind
PCT/AU96/00149	3/15/1996	WO

ANTIGEN COMPOSITION AGAINST MYCOPLASMA

Information

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Date Filed

Date Published

Inventors

CPC

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Abstract

Description

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Priority Claims (1)

PCT Information