WO1996034884A2

WO1996034884A2 - Synthetic peptides of m. gallisepticum, antibodies thereto, their preparation and use

Info

Publication number: WO1996034884A2
Application number: PCT/SI1996/000011
Authority: WO
Inventors: Janko Kos; Dus^¿an BENC^¿INA; Dus^¿an DOVC^¿; Franc GUBENS^¿EK
Original assignee: Institut 'joz^¿Ef Stefan'
Priority date: 1995-05-05
Filing date: 1996-04-29
Publication date: 1996-11-07
Also published as: GB2314560A; SI9500155A; WO1996034884A3; DE19681356T1; AU5414096A; GB2314560A8; GB2314560B; GB9723289D0

Abstract

The invention describes novel synthetic peptides imitating natural epitopes on the membrane of pathogenic mycoplasma M. gallisepticum and a process for the preparation thereof. By the mentioned peptides native antigens in serological tests can be substituted and they can be used as vaccines or as antigens for the preparation of antibodies. Antibodies prepared by means of these peptides recognize native mycoplasma epitopes and are useful in various diagnostic tests for determination of poultry infections with M. gallisepticum, they prevent the growth of the mentioned mycoplasma and are also suitable for the isolation of native or recombinant main immunogenic protein pMGA or its subunits.

Description

Synthetic peptides of M. Gal l i septicum, antibodies thereto, their preparation and use

IPC: A 61 K 39/00

Technical Field of the Invention

The present invention reaches into fields of biochemistry, immunochemistiy and veterinary medicine. It relates to novel synthetic peptides derived from amino acid sequence of single regions of pMGA or its multigenic variant pMGA 1.2, the main immunogenic protein at M. gallisepticum, to a process for the preparation thereof, to antibodies against these peptides and to the use of these antibodies and peptides in diagnostic tests and vaccines in diagnostics and therapy of poultry infected with Mycoplasma gallisepticum.

Technical Problem

In the breeding of poultry such as hens and turkeys Mycoplasma gallisepticum causes great economic damage. Beside direct damage due to deaths, lower production and poor quality of poultry products, there are also high indirect costs comprising the costs of treatment, possible vaccination and obligatory diagnostic examination. It is still a problem to diagnose poultry infections with M. gallisepticum since the processes are unspetific, time-consuming and expensive.

Prior Art

The most reliable method for diagnosing the disease - infection with M. gallisepticum - is the method of isolation and identification of M. gallisepticum (Yoder, H.W., (1991), Mycoplasma gallisepticum infection. In: Diseases of Poultry (B.W. Calnek, HJ. Barnes, C.W. Beard, W.M. Reid and H.W. Yoder eds.) pp. 198-212, Iowa State University Press), which, however, can only be performed by highly specialized laboratories. Additionally, the isolation of M. gallisepticum is very time-consuming, expensive and even impossible for several M. gallisepticum strains. For mass testings serological tests e.g. FSA (Fast Serum Agglutination) and HIT (Haemagglutination Inhibiton Test) and recently also immunoenzymatic assays (ELISA) have been used. All these tests have several deficiencies e.g. unspecific interactions, but their major deficiency is that they detect specific antibodies against M. gallisepticum, which does not allow a diagnosis of the infection in the acute stage when the treatment would be the most effective nor a diagnosis of latent infections.

An alternative to these tests would be immunoenzymatic assays detecting antigens of M. gallisepticum. In such a manner the presence of M. gallisepticum could be detected already in the acute stage. For this purpose suitably avid and selective antibodies against immunodominant surface antigens of M. gallisepticum have to be prepared. For immunization native antigens can be used by way of an appropriate treatment of M. gallisepticum cell membranes. Since the amino acid sequence of haemagglutinin of pMGA (Markham, P.F., Glew, M.D., Whithear, K.G., Walker, I.D. (1993), Molecular cloning of a member of the gene family that encodes pMGA, a haemag¬ glutinin of Mycoplasma gallisepticum, Infection and Immunity 61, pp. 903-909), i.e. of the main immunogenic protein on the M. gallisepticum membrane is known, also synthetic or recombinant peptides, which are similar to single segments of pMGA protein, can be used as antigens.

Peptide synthesis is already an established method for various studies of biological activity of protein molecules. Mainly Merrifield technique of synthesis on a solid car¬ rier (Merrifield, R.B. (1963), Automated Synthesis of Peptides, Science 150, 178-184) is used. According to this method, the first amino acid of C-terminal end of a peptide is over the carboxyl group linked to a solid carrier, and on N-terminal end new amino acids are added to the peptide chain. The use of appropriate protecting groups for N-terminal end and side chains of single amino acids and the introduction of less ag¬ gressive methods of peptide bond synthesis has made possible the automation of the process. Synthesized peptides require additional purification since they contain ad¬ mixtures of reagents as well as a certain portion of peptides with incorrect sequence. The most useful method for this purpose is high performance liquid chromatography (HPLC). The composition and purity of peptides is examined by determination of the composition or sequence in the amino acid chain.

Shorter peptides are usually used as haptenes since they only cause a very strong immune response if they are bound to a carrier molecule. As carrier molecules various proteins e.g. bovine serum albumin, ovalbumin or hemocyanin (KLH) can be used. Covalent binding can take place over the -SH group of cysteines, whereat GMBS (gammamaleimido butyriloxy succinimide) is used as heterobifunctional reagent (Geysen, H.M., Bartheling, S.J., Meloen, R.H. (1985), Small peptides induce antibodies with a sequence and structural requirement for binding antigen com¬ parable to antibodies raised against the native protein, Proc. Natl. Acad. Sci. USA 82, 178-182). Also binding over lysine by means of glutaraldehyde or over carboxyl group by building a carbodiimide bond is used (Erlanger, B.F. (1980), The prepara¬ tion of antigenic haptene-carrier conjugates: A survey Meth. Enzymol. 70, 85-104). Conjugated peptides are suspended in complete Freund's adjuvant and sub- cutaneously injected to test animals. After 2, 3, 7, 8, 9 and 10 weeks the process is repeated with booster injections.

Polyclonal antibodies can be isolated from the serum in several ways e.g. by precipitation (ammonium sulphate, caprylic acid), gel filtration, ion-exchange chromatography or affinity chromatography. As the ligand in affinity chromatog- raphy there can be used immobilized antigen or staphylococcus protein A and strep¬ tococcus protein G, which specifically bind immunoglobulins G especially over Fc receptors.

For the preparation of direct immunoenzymatic assays isolated antibodies have to be conjugated with a marker enzyme, which by degradation of an added chromogen makes possible the visualization of the result. As marker enzymes mainly horseradish peroxidase, alkaline phosphatase and beta-galactosidase are used and the methods of covalent binding are similar to those for the preparation of conjugates between peptides and carrier molecules.

Description of Solution to Technical Problem

By means of determination of cDNA sequence and by analysis of partial amino acid sequence of p67 protein purified by immunoaffinity chromatography on an immobi¬ lized Mab, the amino acid sequence of pMGA, which is the main immunogenic protein with Mr 67000 placed in M. gallisepticum membrane, was determined. It has been found that this protein is responsible for haemagglutination as well as for the adhesion of pathogen on epithelial host cells. Studies of related microorganisms - M. pneumoniae, (Jacobs, E., Fuchte, K., Bredt, W. (1987), Amino acid sequence and antigenicity of the amino-terminus of the 168 kDa adherence protein of Mycoplasma pneumoniae, Journal of General Microbiology 133, 2233-2236; Gerstenecker, B., Jacobs, E. (1990), Topological mapping of the Pl-adhesin of Mycoplasma pneumoniae with adherence-inhibiting monoclonal antibodies, Journal of General Microbiology 136, 471-476) have shown that N-terminal end of Pl-adhesin i.e. a protein having a similar function as pMGA, is the exposed part of the molecule so that it stimulates an immune response.

A peptide similar to N-terminal end of pMGA molecule, which would cause an im¬ mune response, could be used for preparation of antibodies and thus for develop¬ ment of direct immunodiagnostic tests for M. gallisepticum, for vaccine preparation and as an antigen (subunit of pMGA) for determination of specific antibodies in poultry such as hens and turkeys.

Thus the present invention relates to synthetic peptides imitating natural epitopes on the membrane of pathogenic mycoplasma Mycoplasma gallisepticum. By the cited peptides the native antigen in serological tests can be substituted, they can be used as vaccines or as antigens for the preparation of antibodies. Antibodies obtained by these peptides recognize native mycoplasma epitopes and are useful in various diag¬ nostic tests for determination of poultry infections with M. gallisepticum (MG), they prevent the growth of the said mycoplasma and are also suitable for isolation of na¬ tive or recombinant main immunogenic protein of pMGA and its multigenic variant pMGA 1.2, respectively, or their subunits.

The first object of the present invention are synthetic peptides characterized in that they imitate single segments of pMGA or pMGA 1.2 molecule and its N-terminal end respectively and are modified by way of binding cysteine at one or both ends of the peptide chain. The binding of cysteine at one or both ends of the peptide chain makes possible an effective binding of peptides to the carrier molecule and enhances their immunogenicity. Since it has been found that also the N-terminal end of pMGA molecule can vary, it is important that there are several of these peptides, that they differ from each other and that they imitate various parts of the N-terminal end.

Particularly, the present invention relates to synthetic peptides prepared in the above described manner, which are characterized by the following gene sequences:

PI C-T-T-P-T-P-S-P-A-P-N-P-N-P-P-S-N-C pMGA

P2 C-T-T-P-T-P-N-P-T-P-N-P-N-P-P-S-G-C pMGA 1.2

P3 C-T-T-P-T-P-S-P pMGA (first part)

P4 P-N-P-N-P-P-S-N-C pMGA (last part)

P5 P-S-P-A-P-N-P-N-C pMGA (middle part) A further object of the invention is a process for the preparation of the above syn¬ thetic peptides characterized in that the N-terminal end of pMGA or pMGA 1,2 molecule is modified by binding cysteine at one or both ends of the peptide chain.

The fourth object of the invention are therapeutic and diagnostic agents in poultry in¬ fections with M. gallisepticum containing the above peptides as an active ingredient.

Preferably the present invention relates to vaccines, characterized in that they con¬ tain a single or a combination of the above peptides as an active substance.

The sixth object of the invention is use of the above peptides in various combinations or individually for the preparation of vaccines.

Also novel is the use of the above synthetic peptides for determination of serum (systemic) and local antibodies in infections with . gallisepticum.

The eighth object of the invention are the above cited synthetic peptides for use in diagnostics and therapy of poultry infected with M. gallisepticum.

The ninth object of the invention are novel antibodies or parts thereof, characterized by the feature of recognizing native antigens on M. gallisepticum. They are obtained by a usual immunization of animals with the above peptides.

The tenth object of the invention are vaccines characterized by that they contain the above cited antibodies or parts thereof as an active substance.

A further object of the invention are antibodies or parts thereof as described above for use in in vitro or in vivo growth inhibition of M. gallisepticum.

A further object of the invention is the use of the above cited antibodies or parts thereof for the preparation of immunodiagnostic tests for determination of M. gal¬ lisepticum in poultry or any other samples.

An object of the invention is also the use of the above antibodies in analysis of an- tigenic determinants on pMGA or pMGA 1.2.

Still another object of the invention is the use of antibodies in the preparation and purification of pMGA or pMGA 1.2 or their subunits from fermentation broth or from cell suspensions by affinity chromatography. The antigen prepared in such a manner can be used in diagnostic tests and for vaccine preparation.

The present invention is illustrated by the following nonlimiting example.

Example

By an automatic peptide synthesizer (Applied Biosystems) five peptides of different lengths were prepared corresponding to N-terminal sequences of pMGA or its multi- genic variant pMGA 1.2 (Markham, P.F., Glew, M.D., Brandon, M.R., Walker, I.D., Whithear, K.G. (1992), Characterization of a major haemagglutinin protein from Mycoplasma gallisepticum, Infection and Immunity 60, 3885-3891; Markham, P.F., Glew, M.D., Whithear, K.G., Walker, I.D. (1993), Molecular cloning of a member of the gene family that encodes pMGA, a haemagglutinin of Mycoplasma gallisepticum, Infection and Immunity 61, 903-909.):

PI C-T-T-P-T-P-S-P-A-P-N-P-N-P-P-S-N-C pMGA

P2 C-T-T-P-T-P-N-P-T-P-N-P-N-P-P-S-G-C pMGA 1.2

P3 C-T-T-P-T-P-S-P pMGA (first part)

P4 P-N-P-N-P-P-S-N-C pMGA (last part)

P5 P-S-P-A-P-N-P-N-C pMGA (middle part)

Peptides PI and P2 correspond to the first 17 amino acids in pMGA and pMGA 1.2 genes. They differ in three amino acids: S7 - N7, A9 - T9, N16 - G16. In both peptides on the N-terminal end cysteine was added whereby a better binding to the carrier molecule was made possible. The addition of cysteine also makes possible the build¬ ing of peptide loops linked to the carrier molecule over two thiol groups. In such a manner a greater exposure of the peptides and a better immune response was achieved. Shorter peptides P3, P4 and P5 overlap the first, the last and the middle regions of PI peptide. They make possible a more accurate location of the epitope on the N-terminal end of pMGA or PI peptide. To the peptides P4 and P5 cysteine was added to make possible the binding on a carrier molecule over -SH bond.

In the synthesis HOBT/TBTU/DIEA chemistry was used and for the protection of the N-terminal end F-moc (fluorenylmethoxycarbonyl) group was used (Carpino, L.A. (1987), The 9-fluorenylmethyloxycarbonyl family of base-sensitive amino- protecting groups, Ace. Chem. Res. 20, 401-407). Synthesized peptides were cleaved from a carrier with 96% trifluoroacetic acid. Released protecting groups were caught in anisole and ethanediole and protein was precipitated in diethylether. For peptide analysis and purification high performance reverse phase liquid chromatography (RP HPLC) was applied. Milton Roy, USA apparatus with Nucleosil 300 C18 column by Machery-Nagel, Germany, was used. As the mobile phase CH₃CN/H₂O/0.1% TFA was used. From the peptide fractions the solvent was removed by evaporation on the rotary evaporator (Devarot, Elektromedicina) and then they were lyophilized to dry- ness (Hetossic, Heto Lab, Denmark). The dried peptides were stored at -20°C.

For the preparation of polyclonal antibodies the peptides were first conjugated with GMBS (N-y-maleimido butyryloxy succinimide) over a thiol group of cysteine to hemocyanin (KLH) (Kim, S.J., Hirose, S., Miyazaki, H., Ueno, N., Higashimori, K., Morinaga, N., Kimura, T., Sahakibara, S., Murakami, K. (1985), Identification of plasma inactive renin as prorenin with a site directed antibody, Biochem. Biophys. Res. Commun. 126, 641-645). A suspension of hemocyanin in 65% (NH₄)₂S0₄ was centrifuged for 20 minutes at 10000 RPM. The precipitate was dissolved in 0.1 M phosphate buffer , pH 7.0, and desalted by gel filtration on Sephadex G-25 (PD 10 column, Pharmacia, Sweden). Then KHL (4 mg) in 0.1 M phosphate buffer was added to GMBS (0.7 mg) and stirred for 30 minutes at room temperature. Hemocyanin-GMBS was separated from nonreacted GMBS by further gel filtration on Sephadex G-25. To the eluted KLH-GMBS complex 5 mg of each single peptide dissolved in 0.1 M phosphate buffer, pH 7.5, were added and it was stirred for 3 hours at room temperature. The conjugates were stored at -20°C.

0.1 ml of each conjugate were suspended in an equal volume of a complete Freund's adjuvant and rabbits were intracutaneously immunized with the suspension. After two and three weeks the immunization was repeated with incomplete Freund's ad¬ juvant and after eight, nine and ten weeks with a solution of peptides (0.5 mg) in PBS. Rabbits were bled out one week after the last booster injection. The serums were concentrated to one fourth of the volume on an ultra-filter (Amicon, YM5 membrane) and applied to a protein A-sepharose equilibrated with 0.1 M phosphate buffer, pH 8.3, and 0.3 M NaCl. The bound immunoglobulins were eluted with citrate buffer, the pH value 3.5 was immediately set to 7.5 with 1 M Tris buffer, pH 10.5. For binding horseradish peroxidase to immunoglobulins G, a two-step glutaral- dehyde method was used (Avrameas, S., Ternyck, T. (1971), Immunochemistry 8, 1175-1179). 10 mg of horseradish peroxidase (Sigma, type VI, USA) were dissolved in 0.4 ml of 1.25% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, and it was in- cubated for 20 hours at 22°C. Then 0.6 ml of phosphate buffer were added to the reaction mixture and it was dialysed against 0.1 M carbonate buffer, pH 9.2.

Against the carbonate buffer there were also dialysed 5 ml of rabbit immuno¬ globulins G. After dialysis both solutions were combined and incubated for 20 hours at 4°C. Then the remaining reactive groups were blocked by the addition of 0.1 ml of 0.2 M lysine.

The prepared antibodies and conjugates were examined by the following methods:

1. Indirect dot-immunobinding assay (DIBA^ (Kotani, H., McGarrity, G.J. (1986), Identification of mycoplasma colonies by immunobinding, J. Clin. Microbiology 23, 783-785.

2. Indirect immunoperoxidase assay fllPA^ (Benδina, D., Bradbury, J.M., Varga, Z.S., Dovδ, P., Stipkovits, L. (1992), Variation of immunodominant surface antigens in unclassified mycoplasma strains isolated from chickens, 9th International Congress of the International Organisation for Mycoplasmology, Ames, Iowa, USA, IOM Let¬ ters 2, 130).

3. Inhibition of haemagglutination fIHA\ Inhibition of haemadsorption (IHAD) (Benδina, D., Bradbury, J.M. (1991), Indirect immunoperoxidase assay for the detec¬ tion of antibody in chicken mycoplasma infection, Avian Pathology 20, 113-124).

4. Growth inhibiton (GT) of MG in agar and liquid culture media.

5. Immunoblot flB l (Benδina, D., Kleven, S.H., Elfaki, M.G., Snoj, A., Dovδ, P., Dorrer, D., Russ, I. (1994) Variable expression of epitopes on the surface of Mycoplasma gallisepticum demonstrated with monoclonal antibodies, Avian Pathol¬ ogy 23, 19-36.

6. Fast serum agglutination (FSA).

7. Double gel-diffusion precipitation CGDP).

Peptides (PI, P2, P3, P4, P5) were tested by the following methods:

1. Indirect dot immunobinding assay (DIBA) 2. Indirect immunoperoxidase assay (IIP A) - to neutralize the binding of rabbit anti¬ bodies against PI on colonies of M. gallisepticum.

3. Immunoblot (IBL) - PI bound to KLH.

4. Double gel-diffusion precipitation (GDP) - PI and P2 as antigens.

Assay description

Indirect dot immunobinding assay fDIBA^

On Immobilon P membranes - bands (Millipore) 2-3 μ\ of peptides in concentrations from 10 μ-g/ml to 1 mg/ml were brought. After blockade in a phosphate buffer with 0.5% Tween^® 20 for 30 minutes, the bands were incubated with antibodies against peptides (dilutions 1:100 to 1:10⁴) for 45-60 minutes at room temperature. After washing with PBS-T buffer (0.05% Tween^® 20), 3 x 15 minutes, the bands were in¬ cubated for 40 minutes at room temperature with a conjugate (goat anti-rabbit IgG- HRP, Sigma, USA, A-6154), diluted 1:1000. After washing with 2 x PBS-T 0.05%, lx PBS (pH 7.4), there were added DAB (diamino benzidine), 0.25 mg/ml in 0.03% H-,0₂. When the reaction became sufficiently intensive the bands were washed in dis¬ tilled water and dried. Reaction levels were evaluated with respect to signal inten¬ sities and negative controls (preirnmunization rabbit serum + conjugate). The assay was also performed with conjugated rabbit antibodies against peptides.

As antigens there were tested all peptides (PI - P5), affinity-purified (with Mab 86) pMGA obtained from P.F. Markham, MG lectin (Sigma, USA, L5884), MG p64 in ISCOM obtained from G.Czifra (Czifra, G., Tuboly, T., Sundquist, B.G., Stipkovits, L. (1993), Monoclonal antibodies to Mycoplasma gallisepticum proteins, Avian Dis¬ eases 37, 689-696), cell suspensions of MG (1:1000), cell suspensions of M. synoviae strains, M. iowae strains, M. meleagridis (strain 17529) and M. pneumoniae (FH strain).

Long peptides (PI and P2) reacted better than the short ones (P3, P4, P5), and the titers of antibodies (whole serum, IgG without KLH Ab, of M. gallisepticum affinity- purified antibodies) were high (1:10⁴). The antibodies also reacted with pMGA (0.01 mg/ml), M. gallisepticum lectin, p64-ISCOM and with whole cells of various strains of M. gallisepticum (more than 10 strains were tested). In DIBA-test long peptides reacted well with antibodies from serums (and with local antibodies in tracheal mucus of upper respiratory organs) of hens naturally infected with MG.

Indirect immunoperoxidase assay ( IIPA^

IIPA was performed with colonies of 20 various M. gallisepticum strains. The same process as described in the article of Benδina et al. (1994), Avian Path., was used only that in the second step a goat conjugate (goat antirabbit IgG-HRP), diluted 1:200, was used. The titers of antibodies were as follows:

a) whole serum 1:2560 b) IgG (after KHL sepharose) 1:2560 c) affinity-purified antibodies against MG 1:1280 d) control (IgG, bound on KLH) < 1: 20

Positive IIPA reaction could be blocked-neutralized with synthetic peptides PI and P2 in a concentration < 10 μg/ml (incubation with antibodies 30 min., 37°C). Pep¬ tides P3, P4 and P5 were less effective.

Serums of naturally and experimentally infected hens blocked IIPA reaction against peptides PI and P2 with titer 1:8.

Haemagglutination (HA), Inhibition of Haemagglutination (IHA1. Inhibiton of Haemadsorption f IHAD)

IHA and IHAD ability were only shown by the whole serum against PI peptide but not by purified antibodies. IHA tests were performed in microtiter plates (see Benδina et al., (1994), Avian Path.). Clones of various M. gallisepticum and also M. synoviae strains that haemagglutinated hen erythrocytes were used. It was observed that even the whole serum did not give a consistent reaction with all M. gallisepticum clones and strains respectively. However, at least a partial correlation with a strong reaction in IIPA test was shown. It was observed that in some cases no IHA reaction existed at low dilutions of antibodies against PI (e.g. 1:5, 1:10) whereas higher serum dilutions (1:80 - 1:320) gave positive IHA. Such reactions are not unusual since, con- trarily to normal antibodies against M. gallisepticum, the antibodies against PI pep¬ tide are "narrowly specific" whereas in HA reaction certainly several factors and at least two different receptors on erythrocytes take part. In IHAD positive reactions were observed when the whole serum was diluted up to 1:10 (inhibiton of haemadsorption of erythrocytes on M. gallisepticum colonies). Also in this test inconsistent reactions are possible if a strain possesses a HAD property but does not have epitopes for antibodies against PI.

Growth inhibition (GI)

The whole serum against PI peptide inhibited the growth of M. gallisepticum positive clones in GI test. The inhibition zone on agar cultures was 2-4 mm (with 50 μλ of antiserum). Antibodies in the serum inhibited the growth of M. gallisepticum clones with expressed surface epitopes, which means that they are positive in IIPA and that t _*hey react with p67 protein in IBL.

In one case - AAY30 strain - the antiserum against PI peptide partly (1-2 mm) also inhibited the growth of M. synoviae whereas such an inhibition was not observed in other Λf. synoviae strains.

In cases of positive GI there was observed a positive precipitation line pointing to pMGA precipitation (probably of soluble p67 protein released into the medium by M. gallisepticum colonies) with antibodies against PI. This points to a possibility of preparing pMGA by immunoprecipitation by means of antibodies against Pi pep¬ tide.

Immunoblots (IBL)

IBL assays were performed by PHAST-SYSTEM apparatus (Pharmacia, Sweden). The antibodies against PI peptide recognized an epitope on M. gallisepticum p67, which was connected to surface expression on colonies (correlation with IIPA reactivity). The epitope was determined in various M. gallisepticum strains (also in a variant strain K 703) but in no case IBL reactions with other species of mycoplasmas, e.g. M. synoviae, M. iowae or M. pneumoniae took place. Surface proteolysis with en- doprotease of V8 St. aureus (Glu-C, Sigma, USA) of whole cells confirmed that an epitope for antibodies of PI is placed on the N-terminal end of p67. M. gallisepticum strains that did not infect experimental chickens (ALEX, A5969, clone K4/4) did not show any reaction with p67 in IBL either. On the other hand, the strains that promptly infected chickens (S6 Lp and TS 102) reacted with p67 in IBL. Partially, IBL reaction with p67 was also connected with the ability of M. gallisepticum strains to haemagglutinate hen erythrocytes and therefore HA negative clones of M. gal- lisepticum strains F (K9/4/16) and A5969 (K4/4) did not have a reaction with p67 in IBL either. A strong positive correlation exists between recognizing M. gallisepticum p67 antigen with anti-Pi antibodies and with antibodies of infected hens. Clones of M. gallisepticum strains (S6, X95, R), whose growth (GI test) was inhibited by anti¬ bodies against PI, have reaction with p67 in IBL, whereas those clones of M gallisep¬ ticum strains that were not inhibited by antibodies against PI did not react with p67 in IBL either. In IBL assays antibodies in dilutions of 1:1000 were used.

Properties of synthetic peptides

PI (Cl - N17 + C) imitates well the conformation of the epitope located at the N-terminal end of pMGA and at native M. gallisepticum p67 (haemagglutinin). Rab¬ bit antibodies against PI show that this is a variable surface epitope (IIPA) closely connected with p67 (IBL). With less than 10 μg/ml of PI (and P2) it is possible to neutralize the reaction of antibodies against PI in IIPA test (30 min., 37°C). The serum and local (upper respiratory organs) hen antibodies, which were experimen¬ tally and naturally infected with M. gallisepticum, in DIBA reacted well with PI and P2 also at relatively high serum dilutions (1:100 - 1:500).

In IIPA, hen serums block the binding of rabbit antibodies to colonies of M. gallisep¬ ticum and already in an early stage of the infection (or as soon as specific antibodies against MG occur). This points to the potential of PI and P2 to be used as antigens (for serology) or as narrowly defined (subunit) vaccines.

Properties of antibodies

Rabbit antibodies against PI recognize the variable epitope on the surface of membranes of M. gallisepticum (pMGA). Therefore the antibodies react in tests where the antibodies are bound to surface antigenic determinants e.g. DIBA, IIPA, and the whole serum also reacts in GI and IHA and precipitates soluble MG p67.

The antibodies are suitable for analysis of M. gallisepticum cultures with respect to the expression of immunodominant surface epitopes (with IIPA and IBL), which are important in the preparation of M. gallisepticum antigens and M. gallisepticum vac¬ cines. In our analysis all M. gallisepticum vaccine strains used in USA (F strain K810 and 6/85) or in Australia and USA (TS 11 strain) and live vaccine strains of M. gal¬ lisepticum (temperature-sensitive mutants of S6 strain i.e. TS 100 and TS 102 strains) had a variable surface epitope on p67, which was recognized by the mentioned anti¬ bodies. The mentioned live M. gallisepticum strains available on the market are used for vaccination of broilers and layers.

In tests antibodies against the longer PI peptide show a much stronger reaction with surface epitopes of M. gallisepticum than antibodies against shorter P3, P4 and P5 peptides.

After binding antibodies to protein A covalently bound on CNBr-activated sepharose (Pharmacia, Sweden) and after cross-linking with DMP (dimethyl pimelimidate, Pierce, USA), pMGA or its fragments can be isolated from a fermentation broth or from cell suspensions by means of affinity chromatography on a column so prepared.

Claims

1. Synthetic peptides, characterized in that they imitate single segments of pMGA or pMGA 1.2 molecule and are modified by way of binding cysteine at one or both ends of the peptide chain.

2. Peptides according to claim 1, characterized in that they comprise the following peptide sequences:

C-T-T-P-T-P-S-P-A-P-N-P-N-P-P-S-N-C pMGA

C-T-T-P-T-P-N-P-T-P-N-P-N-P-P-S-G-C pMGA 1.2

C-T-T-P-T-P-S-P pMGA (first part) p.N-P-N-P-P-S-N-C pMGA (last part)

P-S-P-A-P-N-P-N-C pMGA (middle part)

3. Process for the preparation of peptides according to claim 1 or 2, characterized in that single segments of pMGA or pMGA 1.2 molecule are modified by bind¬ ing cysteine at one or both ends of the peptide chain.

4. Diagnostic and therapeutic agents in poultry infections with M. gallisepticum, characterized in that they contain peptides according to claim 1 or 2 as an active substance.

5. Vaccines, characterized in that they contain single peptides according to claim 1 or 2 or a combination thereof as an active substance.

6. Peptides according to claim 1 or 2 for use in diagnostics and therapy of poultry infected with M. gallisepticum.

7. Use of peptides according to claim 1 or 2 for determination of serum and local antibodies in infections with M. gallisepticum.

8. Use of peptides according to claim 1 or 2 for the preparation of agents for deter¬ mination of serum and local antibodies in infections with M. gallispeticum.

9. Use of peptides according to claim 1 or 2 in various combinations or individually for the preparation of vaccines. 10. Antibodies or parts thereof obtained against peptides according to claim 1 or 2, characterized in that they recognize native antigens on . gallisepticum.

11. Vaccines characterized in that they contain the antibodies or parts thereof ac¬ cording to claim 9 as an active ingredient.

12. Antibodies or parts thereof according to claim 9 for use in in vitro or in vivo growth inhibition of M. gallisepticum.

12. Use of antibodies or parts thereof according to claim 9 for the preparation of immunodiagnostic tests for determination of M. gallisepticum in poultry or any other samples.

14. Use of antibodies or parts thereof according to claim 9 in analysis of antigenic determinants on pMGA or pMGA 1.2.

15. Use of antibodies or parts thereof according to claim 9 in preparation and purification of pMGA or pMGA 1.2 or their subunits by means of affinity chromatography.

16. Use of antibodies or parts thereof according to claim 9 for vaccine preparation.