WO2004101795A1

WO2004101795A1 - Variable proteins of mycoplasma mycoides, vaccines and process thereof

Info

Publication number: WO2004101795A1
Application number: PCT/SE2004/000753
Authority: WO
Inventors: Joakim Westberg; Anja Persson; Mathias Uhlén; Karl-Erik Johansson
Original assignee: Joakim Westberg; Anja Persson; Uhlen Mathias; Karl-Erik Johansson
Priority date: 2003-05-16
Filing date: 2004-05-14
Publication date: 2004-11-25
Also published as: SE0301436D0

Abstract

The invention relates to novel protein and nucleic acid sequences of variable surface proteins of Mycoplasma mycoides subsp. mycoides biotype small colony which causes Contagious bovine pleuropneumonia. In addition the invention relates to vaccine compositions comprising the proteins of the invention and methods for de-tecting the presence of the bacterium in diagnostic and epidemiological methods.

Description

New proteins

Technical field

The present invention relates to the field of development of vaccines and diagnostic methods for combating Mycoplasma mycoides subsp. mycoides biotype small colony (MmymySC) which causes Contagious bovine pleuropneumonia. In particular the invention relates to novel protein and nucleic acid sequences for variable surface proteins that are useful for the object of the present invention.

Background

Contagious bovine pleuropneumonia (CBPP) is the infectious disease that kills the largest number of cattle in Africa each year. It is a highly contagious respiratory disease, which is caused by Mycoplasma mycoides subsp. mycoides biotype small colony (MmymySC). CBPP is the only bacterial disease included in the A-list of the World Organisation for Animal Health (http://www.oie.int) of prioritised communicable animal diseases, together with fourteen viral diseases. Thus, from a global socio-economic perspective, it is the most important bacterial epizootic. CBPP also affects buffalo and can appear in different forms, ranging from hyperacute and acute variants with high mortality (up to 70%), to subacute and chronical forms with high risk of transmitting the infectious agent from symptomless carriers. The clinical symptoms of acute CBPP involve respiratory distress, cough, cessation of rumination, anorexia, and severe pleuritic pain. CBPP is mainly present in Africa south of Sahara and it is also assumed to be prevalent in Asia. During the 1980's and 1990's, there have also been several outbreaks of CBPP in southern Europe.

MmymySC is a member of the class Mollicutes (trivial name, mollicutes), which have evolved from the Gram-positive bacteria that possess genomes with low G+C content (phylum Firmicutes), and belongs to the genus Mycoplasma (trivial name, mycoplasmas). Mollicutes lack cell wall and are known as the smallest self- replicating organisms. According to phylogenetic studies of the 16S rRNA gene, MmymySC belongs to the Mycoplasma mycoides cluster of the spiroplasma group (1, (2). Five sequenced genomes of mollicutes have been published to date. Mycoplasma genitalium (3), Mycoplasma pneumoniae (4), Ureaplasma parvum (5) (formerly Ureaplasma urealyticum (6)), and Mycoplasma penetrans (7), which all belong to the pneumoniae group, and Mycoplasma pulmonis (8) that belongs to the hominis group (1). The relationship between MmymySC (and other members of the spiroplasma group) and the other sequenced mollicutes is rather distant, i.e. 75-80% similarity as judged from 16S rRNA sequences.

Mycoplasmas are known to express surface proteins that can undergo reversible changes which alter the bacterial cell's antigenic composition. These so called variable surface proteins are assumed to enhance colonisation and adaptation to variable environments during infection. Also, the variation of surface antigens help the bacteria to escape the host immune system and is a major problem in the development of diagnostic tools and vaccines for prevention of CBPP The expression of variable surface protein genes can be regulated either on the transcriptional or the transla- tional level. Transcriptional regulation includes changing the structure of the promoter region, which will lead to either expression of the corresponding protein or no expression at all, so-called on and off regulation or phase variation. The variable promoter comprises a mono- or dinucleotide repeat in the spacer region and during replication, the DNA polymerase sometimes fails to incorporate the correct number of repeats due to polymerase slippage. Translational regulation includes changing the DNA sequence of the coding part of the gene by deletion or insertion of one or several nucleotides, which will lead to a frameshift generating truncated ORFs. The nucleotide insertion and deletion occur at mono- or dinucleotide repeats with the same mechanism as for the transcriptional regulation. Alternative ways for mycoplasmas to vary their expression of surface proteins are also known (Persson, A.M., Doctoral thesis, "Molecular characterisation of Mycoplasma mycoiodes SC", 2002). It is difficult to control CBPP for many reasons. Firstly the clinical symptoms of the disease can be vague and the incubation period of the disease can be very long (more than 200 days in some cases) and therefore infected animals can transmit the disease to a large number of animals before the actual disease breaks out. Currently the available diagnostic tests based on serology are rather insensitive and recently developed molecular diagnostic tools need further development. Secondly, antibiotic treatment can be used as a cure, but has the drawback of increasing the number of chronic carriers of the disease. Therefore, today, the only option to combat the disease with success seems to be extensive vaccination. However, the currently used vaccines are based on live strains of MmymySC and the efficacy of the vaccine depends on the titre and viability of the vaccine. Also, the currently available vaccines require annual re- vaccination in order for the preventive effect to sustain. In WO 01/40471 a lipoprotein of MmymySC was sequenced and used for the development of immunological detection methods of the bacterium and the use of this protein for vaccination was suggested.

However, a key problem with the development of diagnostic tools and vaccine against CBPP are the variable surface proteins discussed above. A diagnostic tool developed to recognise a specific surface protein, will only detect the bacterial cells as long as the epitopes of that protein, which are recognised by the method, is expressed. Similarly, a vaccine will only be efficient against the bacterial cells as long as the cells express the proteins and epitopes that the immune system has developed antibodies against. Since many diagnostic methods and vaccines are developed against surface proteins, the variable expression of many of those in MmymySC is a major problem.

In conclusion, therefore, there is still a need for improved diagnostic tools and vaccines in order to be able to combat CBPP infections. Summary of the invention

The present invention pertains to novel protein- and nucleic acid sequences of variable surface proteins from Mycoplasma mycoides subsp. mycoides biotype small colony, the causative agent for Contagious bovine pleuropneumonia. Knowledge of these sequences is important in achieving control of the disease. The invention also pertains to recombinant vectors comprising nucleic acid sequences of the present invention and host cells comprising such vectors. Vaccine compositions comprising proteins (and/or nucleic acids) of the invention are also contemplated. In addition, the invention is directed to using the protein and nucleic acid sequences of the invention in methods for dectecting the bacterium for use in diagnosis of the disease and epidemiology studies. The invention also relates to a method for producing the proteins of the invention.

Figures

Fig. 1. Codon usage of MmymySC. The data for each genetic codon are displayed in three columns. Column one: the amino acid, column two: the genetic codon, column three: the absolute number of codons in the genome.

Fig. 2. Shows the nucleic acid sequence for promoters of the variable proteins of the present invention.

Definitions

By "variable protein" is meant a protein whose structure, existence and/or amino acid composition can vary between mother and daughter cells and whose alteration is reversible throughout the evolution of an isolate. By "lipoprotein" is meant a complex of a lipid molecule linked to the amino- terminal end of a protein. The lipid part of a lipoprotein anchors the protein to the cell membrane in the bacterium and is composed of a hydrophobic component such as fats, triacylglycerols, fatty acids, glycolipids or phospholipids.

By "surface protein" is meant a protein of which at least part of the protein is located on the surface of a cell in its natural environment.

Whether an antibody "specifically binds" to an antigen can be determined by any commonly known method such as ELISA, Western blot, biosensor analysis and im- munohistology.

Detailed description of the invention

The present inventors have now sequenced the genome of the bacterium responsible for causing CBPP in cattle, Mycoplasma mycoides subsp. mycoides biotype small colony , hereafter called MmymySC. Knowledge of the sequence of this bacterial genome will aid in the development of methods for prevention and treatment of CBPP, and the development of diagnostic tools. The sequencing of the MmymySC genome posed some specific problems that had to be overcome in order for the sequence to be obtained. The MmymySC genome is constituted of about one third of repetitive sequences and is, thus, one of the most redundant genomes that have been sequenced. Thirteen percent of the genome consists of three kinds of insertion sequences (IS) that are present in a large number of copies. Large DNA segments of 12, 13 and 24 kbp in size are also present in the genome as tandem repeats with high nucleotide sequence similarities (>98%) between the copies. Technically speaking, the high copy-number of the IS-elements, the large sizes of the long repeats and the high sequence similarities between their copies caused problems with the genome assembly. In order to solve these problems, two kinds of strategies were performed. Firstly, each IS-copy was isolated within plasmid clones or PCR amplicons and were subsequently sequenced by primer-walking. Since incompletely extended primers in the PCR could hybridise to incorrect IS-copies and cause false positives, the PCR procedures were thoroughly optimised with long extension times and high annealing temperatures. Secondly, the long repeats were sequenced by primer-walking on individual clones, which contained several positions of genome polymorphisms, covering different parts of the repetitive regions. The number of tandem repeats was determined by pulse-field gel electrophoresis of restriction fragments containing the entire repetitive region. The present invention particularly relates to surface proteins of MmymySC, their corresponding genes and analogs and fragments of these.

In a first embodiment the invention relates to variable proteins of Mycoplasma mycoides subsp. mycoides biotype small colony chosen from:

(a) proteins chosen from the group comprising SEQ ID No 22-23 and 26-42;

(b) fragments having at least 20 consecutive amino acids in common with the proteins of (a); or

(c) analogues or recombinant variants showing at least 50 %, preferably at least 70 %, more preferably at least 90 %, most preferably at least 98 % homology to the proteins of (a).

Analogues of the proteins described above can be substitutional, deletional and/or insertional variants of the proteins, which are well known for the person skilled in the art. Recombinant variants of the proteins can also show such variations of the original sequences.

Since these proteins are expressed on the surface of MmymySC, the causative agent of CBPP, they are suitable targets for the development of preventive methods, treatments and diagnostic tools for studies of the bacterium and the disease it causes. The invention also relates to nucleic acid molecules encoding the variable proteins described above including fragments, analogues and recombinant variants thereof. In particular, the invention relates to nucleic acid molecules, chosen from the group comprising SEQ ID No 1-2 and 5-21. In a second embodiment, the invention relates to a variable protein of Mycoplasma mycoides subsp. mycoides biotype small colony chosen from:

(a) proteins chosen from the group comprising SEQ ID No 22-23 and 25-42.

(c) analogues or recombinant variants showing at least 70 %, preferably at least 90 %, and more preferably at least 98 % homology to the proteins of (a); for use in a vaccine.

The invention relates to proteins of MmymySC that show a variable expression at the surface of the cell, here also called variable surface proteins. The expression of these proteins can be varied either by phase variation, that simply turns the genes on and off, as is the case for the proteins of SEQ ID No 22-23 and 25-36. This variation is caused by a variation of the number of bases in the spacer region between the -35 and -10 regions of a bacterial promoter. The only previously reported gene to be involved in variation of surface proteins in MmymySC is vmm (32) (MSC_0390) (SEQ ID No 3; corresponding protein sequences is shown in SEQ ID No 24), which encodes a phase variable protein precursor (Persson et al., 2002, J Bacteriol, 184:3712- 3722). The expression of Vmm can be switched on and off by alternating the number of TA-repeats in the promoter spacer. Also, from Thiacourt et al. (Veterinary Microbiology, 200; 72(3-4):251-68), disclosing a phylogeny study, a sequence cor- repsonding to SEQ ID No 25 is known. In the present invention, five additional genes encoding proproteins which have promoters with five to twelve TA-repeats in the promoter were identified (SEQ ID No 1, 2, 4, 5, 6; corresponding to protein sequences 22, 23, 25, 26, and 27, respectively). The DNA sequence assembly contains clones with different numbers of TA repeats in the promoters of MSC_0117 and MSC_1005, suggesting that polymerase slippage during replication does occur in these promoters. Furthermore, there are homonucleotide regions consisting of 15 to 23 As or Ts, which are located in the putative promoter of nine MmymySC surface protein genes (SEQ ID No 7-15). Again, these repetitive sequences may be involved in transcriptional control. An alternative mechanism for varying the expression of surface proteins was also identified in two surface protein genes according to SEQ ID No 16-21. These contain a mononucleotide stretch of 10 to 14 As or Ts within the gene per se. These repetitive sequences may lead to size and amino acid variation of the resulting proteins due to frameshift caused by misincorporation of the correct number of repeat residues. In total, there are 67 proprotein genes and 346 transmembrane protein genes in the genome whose products are potential virulence factors as they may be involved in adherence or host cell interactions. No function has yet been assigned to the variable protein according to SEQ ID No 37; the corresponding nucleic acid sequence is shown in SEQ ID No 16.

The invention also relates to recombinant vectors comprising a nucleic acid molecule as described above. Examples of vectors suitable for the present invention include pAFFδc, pBADlδ, and different pET vectors, such as pET28, when Escher- ichia coli is used as a host. Lartigue et al (personal communication) have recently developed a plasmid that was shown to replicate in MmymySC which could be used in the present invention. Mycoplasmas use the codon UGA to express the amino acid tryptophan, instead of using it as a stop codon that is common in most other bacteria. Therefore the codon UGA has to be modified before use in other organisms than Mycoplasma in order for the amino acid tryptophan to be expressed in these organisms.

The codon usage of MmymySC is shown i Fig. 1. The data for each genetic codon are displayed in three columns. Column one: the amino acid, column two: the genetic codon, column three: the absolute number of codons in the genome.

Also included in the present invention are host cells comprising such vectors comprising the nucleic acid sequences according to the present invention. The vector optionally also contains sequences for regulating the expression of the proteins en- coded by the nucleic acid sequence and sequences for selecting the presence of the vector in a host cell, such as genes encoding antibiotic or heavy metal resistance or any other suitable selection marker. The choice of suitable host cells, regulatory sequences, selection markers are within the skills of a person skilled in the art. Suitable host cells include both Gram positive bacteria such as Staphylococci, Streptococci, Lactococci, and Gram negative bacteria such as Escherichia coli and Salmonella.

Also included in the present invention are DNA libraries comprising nucleic acid sequences of the proteins according to the present invention.

Also included in the present invention is a fusion protein, comprising at least two variable proteins of the invention and at least one linker molecule positioned between the proteins. Hereby, a polyvalent vaccine against MmymySC is provided.

The background for this aspect is as follows: A vaccine containing a fusion protein including proteins, represented by the sequence of SEQ ID No 22-42 or parts thereof, has several advantages over a vaccine containing a cocktail of the same proteins, (i) It enhances the immunogenicity of the vaccine and the avidity between the antibody and the vaccine, (ii) It facilitates the production of the vaccine since fewer proteins have to be expressed and purified, (iii) It contributes to a more constant ratio between the amounts of antigen epitopes in the vaccine.

The basic idea of producing a fusion protein is to fuse two or more genes or parts of the genes, represented by the sequence of SEQ ID No 1-21 excluding the stop co- dons, with a linker (hinge) between each gene. The fusion gene must not contain any stop codons except at the 3 '-end. The linker is designed to enable the native folding of the individual protein fragments in order to mimic the MmymySC antigens in the best way. The linker can also contain a functional protein, like for exam- pie T-cell epitopes, to enhance the immune response and also a multiple cloning site, to be able to add, delete or substitute gene fragments in the recombinant gene.

The fusion protein of this aspect of the invention comprises two or more proteins of the invention, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more proteins of the invention. Advantageously, the fusion protein comprises a number of proteins that is sufficient in order for the fusion protein to provide a satisfactory vaccine effect in itself. In a preferred embodiment, the fusion protein comprises more than two proteins of the invention, and even more preferably, the fusion protein comprises more than five proteins of the invention.

There are different procedures that can be used for the production of the fusion protein. For example, in order to build a fusion protein, Pan et al. (Pan et al, J Immunol. 2004;172(10):6167-74) synthesised 80-mer oligos, representing overlapping fragments of the gene coding for the fusion protein. These oligos were then used in an asymmetric PCR to obtain the full-length fusion protein. In this case, the linker consisted of two glycine-prolin-glycine amino acid repeat motifs, which were flanking a multiple cloning site. The fusion gene was inserted into a pBSK plasmid via. Xhol and Eco I restriction sites. The recombinant plasmid was transferred into Escherichia coli DH5α and several clones were sequenced to identify a clone with error-free sequence. A second procedure is to use already available genes, represented by the sequence of SEQ ID No 1-21, and linkers that overlap the genes with about 10-30 nucleotides in an asymmetric PCR and thereafter continue as demonstrated by Pan et al. (Pan et al., 2004). A third procedure is to use linkers that are li- gated to the genes and thereafter continue as demonstrated by Pan et al. (Pan et al., 2004). However, as the skilled person easily realises, the invention is not restricted to these examples, and other methods for producing a fusion protein according to the invention are also included in the scope of the invention. In one embodiment the present invention relates to a vaccine composition comprising at least one protein according to the present invention. The vaccine composition preferably comprises at least one protein that has at least 60 amino acids, preferably at least 80 amino acids, more preferably at least 100 amino acids, most preferably at least 150 amino acids in order to enhance the immune response of the vaccine composition. Preferably a longer polypeptide chain is used, since longer polypeptides have more antigenic sites to which are targets for the immune system in the development of antibodies, i.e. longer polypeptides are more immunodominant. The use of pure protein preparations for vaccine purposes has several advantages over whole cell vaccines. Firstly, it is possible to produce a vaccine, which has a defined amount, composition and purity of the active substance. Secondly, it is easier to store and transport protein-based vaccines. This is specially an advantage in respect of live whole cell vaccines, which can be very sensitive to storage and transport conditions. Thirdly, when live or attenuated vaccines are used, there is always a risk that the vaccines cause outbreak of disease. In addition, to date, use of cell lysates of MmymySC for vaccination purposes has not been successful. Therefore the knowledge of the sequence of surface proteins of the present invention has the advantage of allowing the development of new and better vaccine compositions in order to prevent CBPP.

One embodiment of the present invention is directed to the use of at least one protein according to the present invention for the preparation of a vaccine composition for use in preventing disease caused by Mycoplasma capricolum, gallisepticum, ge- nitalium, hominis, hyopneumoniae, mycoides, pneumoniae, specifically Mycoplasma mycoides.

Another embodiment of the present invention is directed to the use of at least one protein according to the present invention for diagnosing the occurrence of Mycoplasma capricolum, gallisepticum, genitalium, hominis, hyopneumoniae, mycoides, pneumoniae, specifically Mycoplasma mycoides. Host cells comprising nucleic acid sequences, preferably inserted into a suitable vector according to the present invention, which allows the host cell to express the proteins of the invention can also be used directly, in a live, attenuated or killed form, to deliver the antigen to the subject to be immunised. The use of whole cells for vaccination has the advantage that the antigenic proteins need not to be purified from the cells. Also, if a non-pathogenic host cell, such as Staphylococcus carnosus, Staphylococcus xylosus andE. coli, is used in a live form to deliver the antigens, the dosage of cells in the vaccination is not very critical, since there is no risk of disease, compared to when live vaccines of the pathogenic strain is used. This faciliates storage and transport of the vaccine compositions. Another advantage with the use of live, non-pathogenic host cells expressing the antigens is that they may have an ability to colonise the body of the subject immunised, which prolonges the time the antigen is present in the body which increases the chance for successful vaccination. The cells may also in themselves act as adjuvants. Alternatively, viruses and phagemides comprising nucleic acid sequences encoding the proteins of the invention can be used for vaccination So called naked DNA can also be used for vaccination purposes. This involves administration of nucleic acids according to the present invention inserted into a vector such as a plasmid alone. Vaccine compositions comprising combinations of protein and nucleic acids according to the present invention can also be used in a vaccination regime.

The vaccine composition may also optionally comprise additional components, such as vaccine adjuvants, carriers and auxiliary substances. Adjuvants are substances that are known to stimulate the immune system of a vaccinated animal. Adjuvant suitable for the vaccine composition of the present invention include, but are not limited to, Freund's complete adjuvant, Marcol 52:Montanide 888 (Marcol is a trademark of Esso, Montanide is atrademark of SEPPIC, Paris), squalane or squa- lene, Adjuvant 65 (which contains peanut oil, mannide monooleate and aliminium monostearate), mineral gels such as aliminium hydroxide, aluminium phosphate cal- cium phosphate and alum, surfactants such as hexadecylamine, octadecylamine, ly- solecithin, dimethyldioctadecylammonium bromide, N,N-dioctadecyl-N,N'-bis(2- hydroxyethyl)-propanediamine, methoxyhexadecylglycerol and pluronic acids, poly- anions such as pyran, dextran sulphate, polyacrylic acid and carbopol, peptides and amino acids auch as muramyl dipeptide, dimethylglycine, tuftsin, trehalose dimyco- late and Immunostimulating complexes (ISCOMS). The proteins of the present invention can also be administered to a subject incorporated into liposomes or other microcarriers, after conjugation to polysaccharides, proteins or polymers or in combination with Quil-A to form immunostimulating complexes. The vaccine composition is preferably used to immunise cattle in order to prevent contagious bovine pleuropneumonia (CBPP). The administration route that allows an appropriate immune response can be used, including subcutaneous, oral, nasal and rectal administration. The dosage to be administered and the frequency and number of injections which are suitable can be optimised in each specific case by the person skilled in the art.

The variation of surface antigens on MmymySC has, as described above, been a problem in the development of vaccines against CBPP. Since a vaccine based on whole cells will only express one certain array of antigens of all the cells possible arrays of antigens, the vaccine will only be effective as long as the proteins to which antibodies are raised are present. However, as known, the expression of surface antigens is varied, which helps the MmymySC cells to escape host defences. In contrast, with the new knowledge of the protein sequence of the variable proteins according to the present invention it is now possible to use the pure proteins, and not whole cells or cell lysates for vaccination purposes. Vaccine compositions comprising a single protein according to the present invention can be used. However, preferably, the present inventors suggest the use of a mixture of the protein parts of at least two variable proteins, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 proteins, in the vaccine composition according to the invention. Thereby, the problem with variation of surface antigens of MmymySC can be avoided. The use of a mixture of proteins for vaccination purposes has the additional advantage of increasing the spectrum of MmymySC strains that the vaccine is effective against.

The present invention also relates to affinity reagents, such as antibodies and affi- bodies, recognising a protein according to the present invention. The antibodies are prepared using standard vaccination techniques for raising antibodies, which involve administration of at least one protein according to the present invention to an appropriate host, such as a mouse, rat, rabbit, goat etc. Both polyclonal and monoclonal antibodies prepared using standard techniques within the art of antibody production are scope of the present invention.

The proteins, and nucleic acids encoding the proteins, of the present invention can also be used in a method for detecting the presence of MmymySC. Such a method can be used for diagnostic purposes as well as for studies of the epidemiology of CBPP. The diagnostic method can be a method based on the detection of surface proteins of the invention, such as ELISA. Alternatively, Growth Inhibition Test (GIT) can be used for detection of MmymySC. In GIT, after spreading of bacteria on a plate with solid culture medium, a drop or a patch comprising antibodies against the proteins of the present invention is placed on the plate. Thereafter, the bacteria are cultivated at an appropriate temperature. If bacteria are present to which the antibodies can bind, a clear spot appears around the area where the antibodies were applied. Latex agglutination test can be used to test whether an animal is infected with MmymySC. In this test, latex beads are supplied with antigens (such as the proteins of the present invention) to which antibodies from the animal will bind if present in the animal (i.e. if the animal is infected with MmymySC). This will cause agglutination of the beads that can be detected. The method can also be based on the detection of a nucleic acid sequence according to the present invention, for example in PCR-reaction, wherein primers are designed to specifically bind to the nucleic acid sequences according to the present invention. Also, methods employing a DNA-hybridisation technique, such as Southern blotting, wherein nucleic acid probes labelled for example radioactively or by fluorescent markers are designed are used for detecting the presence of nucleic acid sequences according to the present invention. The complement fixation test (CFT) is a method that detects the presence of antibodies against MmymySC. A MtitymySC-spGCϊfic antigen (i.e. protein or nucleic acid of the present invention) is added to the sample together with a suitable source of complement that can be used for the purposes of the present invention. In a positive test, the antibodies in the test serum react with the antigen and activate complement. An indicator such as red blood cells (RBC) is added to detect complement left in solution. Inactive complement will lyse the RBCs. Consequently, A positive test serum absent of inactive complement will render the solution a red colour due to non-lysed RBCs.

The present invention also relates to a kit for detecting MmymySC. Such a kit can be directed to the detection of the proteins of the present invention and can comprise primary antibodies that specifically bind to the proteins of the invention. In addition, such a kit would comprise secondary antibodies, which are able to bind specifically to the primary antibodies and not to the target cell or protein. Bound to the secondary antibody would be an enzyme such as alkaline phosphatase, peroxidase or ure- ase that can catalyze a reaction that converts a colourless substrate, which also would be included in the kit, into a coloured product. A kit for detecting MmymySC can also be directed to detection of nucleic acid sequences according to the present invention. A kit for performing a PCR reaction would comprise, contained in different vessels, in addition to primers that bind to nucleic acid sequences according to the present invention, a DNA-polymerase, such as Taq-polymerase, and dNTPs.

Promoter sequences which cause variation of the expression of surface proteins of the invention (as discussed above) are shown in Fig. 2. Determination of the number of nucleotide repeats in the promoter are useful for, for exemple, diagnostic purposes. Since the number of repeates determines whether the expression of the protein is switched on of off, deteπnination of the number of repeats in the promoter allows establishment of if the protein is expressed in a certain population of cells, which for exemple can be useful in clinical tests. Also, determination of the number of repeats in the promoter can be used in epidemiological studies in distinguishing between different strains of MmymySC.

One embodiment of the present invention is related to a method for preparing a protein of the invention, which methods comprises the steps of: a) introducing a vector comprising a nucleic acid sequence encoding a protein of the present invention as described above into a suitable host cell (for suitable host cells, see above) b) culturing the host cell under conditions that allows the expression of said protein c) isolating the protein of step b) from the host cell or cell culture supernatant.

Even though the invention primarily is directed to vaccine compositions, diagnostic tools etc. for MmymySC, it can also be used for other Mycoplasmas, such as Mycoplasma adleri, agalactiae, agassizii, alkalescens, arginini, arthritidis, auris, bovi- genitalium, bovine group 7, bovirhinis, bovis, bovoculi, buccale, californicum, ca- nadense, canis, capricolum, caviae, collis, conjunctivae, coragypsi, cottewii, croco- dyli, edwardii, faucium, felifaucium, feliminutum, felis, fermentans, flocculare, gal- linaceum, gallinarum, gallisepticum, gateae, genitalium, hominis, hyopharyngis, hyopneumoniae, hyorhinis, hyosynoviae, imitans, incognitus, iowae, lacerti, leocap- tivus, leopharyngis, lipophilum, meleagridis, mobile, muris, mycoides, neurolyti- cum, orale, ovipneumoniae, penetrans, PG 50, pirum, pneumoniae, primatum, pul- lorum, pulmonis, putrefaciens, salivarium, simbae, sp., sp. H3110, sp. UC/MF, sturnidae, sualvi, synoviae, testudinis, virus PI, volis, yeatsii, /preferably Mycoplasma capricolum, gallisepticum, genitalium, hominis, hyopneumoniae, mycoides, pneumoniae, even more preferably Mycoplasma mycoides, most preferably MmymySC. The genome of MmymySC consists of a single circular chromosome with a size of 1,211,703 bp and it possesses 985 putative genes including 72 genes located within insertion sequence (IS) elements (see Supplementary Information). Biological roles were assigned for 59% of the genes, 14% are similar to genes with unknown function in other species, and 27% are unassigned genes that are at present unique for MmymySC. The G+C content of 24.0 mol% is the lowest among the bacterial genomes sequenced thus far. Intragenomic sequence comparisons show that MmymySC has a high degree of long repetitive sequences compared to other genomes (see Supplementary Information). In total, the repetitive sequences in MmymySC constitute 29% of the genome. The largest repeats are 24, 13, and 12 kb. They are flanked by IS elements and have been duplicated once in tandem. The paralogous genes, generated in these processes, have been subjects for evolutionary pressure, which have led to truncation of many of them.

More than 13% of the MmymySC genome consists of three kinds of IS-elements and it is thereby the most IS-dense bacterial genome that has been sequenced to date. ISMmy7 (10), which is 1,670 bp long, is present in eight full-length and one truncated copy. ISMmyi-like sequences were also found in the bovine pathogen Mycoplasma bovis while mycoplasmas that are phylogenetically closer to MmymySC lack ISMmy/ (10). This observation suggests horizontal transfer of ISMmy7 between MmymySC and M. bovis. Southern blotting of 15 MmymySC strains with an IS- Mmy7 probe showed a unique hybridisation pattern for the vaccine strain TlSr49, which makes ISMmy/ a potential marker to distinguish the vaccine strain from naturally occurring field strains. The other two IS-elements are IS 1634 (23), which is 1,872 bp long, and IS1296 (24), which has a size of 1,485 bp. IS1634 exists in 60 copies where two copies are split by IS-elements and one is truncated. IS 1296 is present in 28 copies including four that are interrupted by IS-elements and seven truncated copies. The IS-elements are evenly distributed across the genome except for three larger IS- free regions, which are located at positions 285,937 to 363,559, 471,574 to 592,871, and 828,541 to 881,279 There is no obvious explanation for the absence of ISs in these regions except that they partly constitute several conserved regions of the mollicutes, like the operons of the ribosomal protein and the ATP synthase genes, and the pyruvate dehydrogenase gene cluster. Six other transposase-like ORFs were found. One of them (MSC_0603) resemble transposases of the IS30 family and one (MSC_0699) is similar to transposases of the IS3 family. The remaining four are possible remnant transposases of ISMmy/ (MSC_0120 and 0125) and IS1296 (MSC_0213 and 0836). However, no additional characteristic features of an IS- element have been found for these putative transposases.

Usually, the origin of replication (oriC) of a genome can be estimated by calculating the GC-skew, defined as (G-C)/(G+C), where the leading strand normally contains more Gs than Cs. For most bacterial genomes, the GC-skew diagram has two nodes that are located at the origin and the termination of replication. The GC-skew of MmymySC reveals the putative position of the termination of replication, but it does not follow a normal pattern at the expected oriC locus. For most bacterial genomes, the dnaA and dnaN genes are located directly downstream of the oriC. These two genes are situated opposite the putative termination of replication in MmymySC, which suggests that oriC may be located in the vicinity of these genes. This has very recently been confirmed by Lartigue et al., who have produced a DNA plasmid with the dnaA region, obtained from the genome sequence of MmymySC, as the oriC and have shown that it can replicate in MmymySC (personal communication). Anomalous patterns of the GC-skew have previously been shown for Yersinia pestis (25) where ISs were found at the borders of the three regions with deviating patterns. Since the IS-elements in MmymySC are distributed throughout the whole genome, it is difficult to determine their influence on the GC-skew pattern. Notably, the GC- skew for MmymySC follows the direction of transcription, i.e. the transcripts are located on the strand with more Gs than Cs.

Genomes with low G+C content are particularly rich in As and Ts in the third position of their genetic codons. In MmymySC, 91.4 mol% of the nucleotides in the third position are A or T. Strikingly, the genome only possesses ten CGG codons (see Supplementary Information), which is in agreement with the fact that MmymySC is only possessing a single tRNA (tRNA^ACG)) for decoding the CGN codons (where N is A, C, G, or T), whereas the other five sequenced mollicutes have two. Interestingly, the CGG codon has not so far been found in Mycoplasma capricolum, which is a close relative to MmymySC (>99% similiarity between their 16S rRNA genes) and which also belongs to the M. mycoides cluster. It has been experimentally shown that translations of synthetic genes in M capricolum are terminated at the CGG codons, suggesting that the CGG codon is a nonsense codon in M. capricolum (26). The hypothesis was that there has been a strong pressure for AT-biased mutations and that the CGG codons have been converted to synonymous arginine codons. This might be the consequence for lacking the corresponding tRNA. It would be interesting to study if the CGG codon is a nonsense codon also in MmymySC. The universal stop codon UGA is coding for tryptophan in most of the mollicutes. Interestingly, the UGA codon is 24 times as frequent in the MmymySC genome as the synonymous codon UGG. A plausible explanation for the large amount of UGA codons is the evolutionary pressure towards a lower G+C content of the genome. The most abundant amino acids in MmymySC are lysine, isoleucine and leu- cine, which together comprise 31 mol% of all amino acids (see Supplementary Information). In contrast, cysteine occurs only twice per protein on average, thus being the least frequent amino acid. This is in general agreement with other mollicutes.

In spite of large efforts, the mechanisms behind the ability of MmymySC to cause disease are virtually unknown. However, there are some theories that have been ex- perimentally tested. Already in 1976, it was shown that intravenous injection of the capsule from MmymySC in calves evoked pulmonary oedema as in natural lesions of CBPP, suggesting that the capsule has a direct toxic effect (27). There are also some indications that increased capsular content associates with reduced phagocytosis by host cells (28). The MmymySC genome contains two clusters of genes involved in the synthesis of the capsule.The first one is located between positions 127,251 and 130,842 and comprises three genes encoding two putative glycosyltransferases and a UTP-glucose-1 -phosphate uridylyltransferase. The second one is located between positions 1,108,435 and 1,133,176 and consists of a gene coding for UTP-glucose- 1 -phosphate uridylyltransferase and a region that is tandemly repeated twice. Each repeat contains genes encoding two putative glycosyltransferases, a UDP-glucose 4- epimerase, a UDP-galactopyranose mutase, and the ATP-binding component of an oligopeptide-specific ABC transporter. The large cluster is also intergenically interspersed with four IS1634-copies. The redundancy of capsule biosynthesis genes might enable MmymySC to produce a relatively high amount of capsule and thereby increase the virulence of the organism. It might also be a way of varying the composition of the capsule in order to escape the immune system of the host.

African and Australian strains of MmymySC form hydrogen peroxide by oxidising glycerol at high rates, while the European strains do not produce any detectable amounts (29). Production of hydrogen peroxide and other active oxygen containing molecules have been suggested as potential virulence factors of mycoplasmas. Since the European strains are less virulent than the African and Australian strains (30), the formation of hydrogen peroxide is a potential factor of pathogenicity in Mmy- mySC. The strain of present study, MmymySC PG1 and whose origin is not known, contains a cluster of genes (gtsA, gtsB, and gtsC) encoding the ABC transporter proteins involved in glycerol transport. The IppB gene encoding a lipoprotein precursor is located immediately downstream of the glycerol uptake cluster. Presumably, it codes for the glycerol-binding subunit, since the gene encoding the substrate- binding component normally is located in the vicinity of the associated ABC transporter genes and has the structure of a prolipoprotein coding gene. All genes in the glycerol uptake cluster are present in the African and Australian strains but in the European strains gtsB is truncated and gtsC and IppB are absent (31). Both glycerol kinase and gly cerol-3 -phosphate oxidase, which are responsible for the production of hydrogen peroxide from glycerol, are present in MmymySC PG1 .

It is noteworthy that seven ISMmyi-elements have been inserted into promoters with TA-repeats (data not shown), thus abolishing the expression of putatively phase variable proteins. Three of these spliced promoters are located upstream of genes encoding membrane-associated proteins. The other four lack the corresponding genes, which probably have been eliminated from the genome.

The number of genes that belong to the different functional categories in MmymySC is approximately the same as for the other sequenced mollicutes. The large number of transport proteins in MmymySC, compared to the other species but M pulmonis, may result in MmymySC being better equipped to persist different tissue environments, reflecting its capability to form systemic infections. The high number of genes of other categories is because of the large amount of transposases located within the IS-elements.

The set of genes encoding proteins involved in replication, transcription and translation resembles the repertoire of the other sequenced mollicutes. The ribosomal RNA genes are clustered in two rRNA operons with the gene order 16S rRNA-23S rRNA-5S rRNA, which are separated by 586 kbp. The MmymySC genome comprises 30 tRNA genes and their corresponding tRNAs have specificity for all amino acids. A reduced set of tRNAs is common in mollicutes, of which M pulmonis has the smallest set of 29 tRNA genes (8). Mollicutes are known to have a restricted biosynthetic capacity. For instance, they lack a complete tricarboxylic acid cycle, have a scarce ability to synthesise amino acids and are not able to synthesise purine and pyrimidine bases de novo. MmymySC has been shown to metabolise the exogenous sugars glucose, fructose, N- acetylglucosamine, glycerol, 2-oxobutyrate, and pyruvate at moderate concentrations and mannose and L-lactate at high concentrations (33). In contrast, it is not able to utilise maltose and trehalose. All genes of the phosphotransferase systems (PTS) of glucose, fructose and mannitol have been identified. The sugars transported into the cell by these systems are degraded by the enzymes of the Embden- Meyerhof-Parnas (EMP) pathway to pyruvate and subsequently to lactate and ace- tyl-coenzyme A. The deoC gene is present and it is encoding deoxyribose-5- phosphate aldolase, which connects the EMP pathway with the DNA metabolic pathway via 2-deoxyribose-5-phosphate and glyceraldehyde-3 -phosphate. The oxi- dative branch of the pentose phosphate pathway is missing in MmymySC as well as in most other mollicutes except for Acholeplasma species (34). In the nonoxidative branch, only transaldolase is missing, suggesting an alternative route or enzyme for the conversion of sedoheptulose-7-phosphate and glyceraldehyde-3 -phosphate to fructose-6-phosphate and erythrose-4-phosphate.

The biosynthesis of nucleotides in MmymySC follows either of two routes. Firstly, adenine/hypoxanthine-guanine/uracil phosphoribosyltransferase catalyses the formation of nucleoside monophosphate from PRPP and a nucleobase. However, since nucleobases are not synthesised de novo in mollicutes, exogenous nucleobases have to be transported into the cell. Secondly, nucleoside kinases generate nucleoside monophosphates by phosphorylation of nucleosides. In some mollicutes there is an interconversion of deoxynucleoside monophosphates by nucleoside phosphotransferase, but it seems that MmymySC is lacking this actual gene. Experiments made by Wang et al. (35) showed that MmymySC is capable of phosphorylation of all four deoxynucleosides by only two enzymes, thymidine kinase and a deoxyguanosine kinase.

In addition to the PTS transporters mentioned above, eight complete ATP binding cassette (ABC) transporters have been identified in MmymySC. According to in silico analyses, these transporters are capable of transferring sugars, oligopeptides, spermidine and/or putrescine, phosphate, alkylphosphonate, glycerol, and a non- identified solute across the plasma membrane. A unique feature of the sper- midine/putrescine ABC-transporter system is that one of the permease components and the substrate-binding component are encoded by one gene (potCD) in MmymySC. These are normally encoded by two separate genes, potC and potD. The permease and substrate-binding domains of potCD are separated by approximately 350 amino acids, and the signal peptide sequence of the potD genes is missing in the potD-li s part of potCD. There are several additional PTS and ABC transport systems although not all subunits have been identified. The missing components may be among the non-assigned hypothetical proteins.

A minimal gene set for cellular life has been postulated by comparing the genome sequences of M genitalium and Haemophilus influenzae (36). Since these two species belong to different phyla, it was earlier believed that their common genes would be essential for growth, although only two phyla is probably too few. A comparison of the gene set of the minimal genome to the MmymySC gene set showed that 11 out of 254 genes of the minimal genome are absent in MmymySC. Except for the genes encoding the heat shock proteins GroEL and GroES, which are also missing in M pulmonis and U. parvum, the genes coding for three hypothetical proteins (MG055, MG127 and MG143 in M genitalium), the ribosomal protein S6 modification protein (MG012), the cytidine deaminase (MG052), the riboflavin kinase (MG145), the thymidylate synthase (MG227), the dihydrofolate reductase (MG228), and an his- tone-like protein (MG353) are absent. MmymySC is the first bacterium that causes a severe disease in livestock and whose genome has been sequenced. Knowledge of the genome sequence of MmymySC will most likely facilitate the development of new vaccines, drugs and diagnostic tools for CBPP. All combinations of the variable proteins are target candidates for vaccine development. In addition, substances that will inhibit the uptake of glycerol and production of the capsule are potential candidate drugs. Further analyses of the genome may reveal additional pathogenic mechanisms of MmymySC. Since this is the first genome that has been sequenced in the spiroplasma group of the mollicutes, it will serve as a good complement to the five previously published mollicute genomes for the study of the evolution of the mollicutes. The generated data will make it possible to perform functional analyses of the whole proteome of MmymySC by for instance gene knockout and microarray technology. The genome sequence also reveals an ongoing process of large rearrangements of the genome, without any compulsions of preserving the direction of the transcripts.

Examples

Materials and Methods

Example 1. Construction of random libraries. The MmymySC type strain

PG1 was grown in F medium (9). Genomic DNA was prepared and purified by proteinase K lysis and phenol/chloroform extraction. Five kinds of plasmid libraries were created. The A-library was generated by nebulization, the B- and C-libraries by partial poI-restriction and the D- and E-libraries by partial Sau3 Al-restriction of genomic DNA. The size fractions were 0.8-1.2 kbp for the A-library, 2-4.5 kbp for the B- and D-libraries and 4.5-9 kbp for the C- and E-libraries. The DNA fragments of the nebulized library were cloned with S αl-restricted pUC18, the - øl-libraries were cloned with 'coRI-restricted pUC18 and S w3AI-libraries were cloned with -BαmHI-restricted pUC 18. Example 2. DNA sequencing. The plasmid clones of the A-library were prepared for DNA sequencing by PCR and the plasmid clones of the four other libraries were prepared by purification of the plasmids with a plasmid preparation kit from MilliPore™ (Bedford, MA, USA). Both ends of the plasmid inserts were sequenced with BigDye™ Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer, Norwalk, CT, USA) or DYEnamic™ ET terminator cycle sequencing premix kit (Amersham Biosciences, Piscataway, NJ, USA) and the sequencing reactions were loaded on ABI PRISM 3700 (PE Applied Biosystems, Foster City, CA, USA) and MegaBACE 1000 DNA sequencers (Molecular Dynamics, Sunnyvale, CA, USA). The IS-elements and the long DNA repeats were sequenced separately by primer- walking on plasmid clones or PCR amplicons (10). Non-repetitive gap sequences and sequences of poor quality were sequenced directly from genomic DNA (11).

Example 3. Genome restriction map. The MmymySC genome was mapped by two-dimensional pulse field gel electrophoresis (PFGE) of MM and Smal restricted fragments and one-dimensional PFGE of Sail, Aatll, Aviϊl, Pvul, andNcøl restricted fragments. This map and a previously published genome map of MmymySC (12) were used for determination of the accuracy of the genome assembly.

Example 4. Assembly and Genome analysis. Basecalling, vector sequence elimination and assembly of the sequences were performed with PHRED (13) and PHRAP (P. Green, University of Washington; http://www.phrap.org/). The assembly was visualised and edited in the COΝSED program (14). The genome sequence was analysed and annotated with the aid of GEΝDB (15), a flexible open source genome annotation system for prokaryote genomes. Open reading frames (ORFs) were predicted by using GLIMMER 2.0 (16) and searched for homology to sequences of the public databases with BLASTΝ and BLASTP (17). Signal peptide sequences were predicted by SIGΝALP (18) and protein motifs were searched for in the Pfam database (19) by using HMMER (Eddy, S. R. (2001) HMMER: Profile hidden Markov models for biological sequence analysis; http://hmmer.wustl.edu/).

The tRNA genes were identified with tRNAscan-SE (20). Putative transmembrane proteins were identified by TMHMM 2.0 (21). Codon usage was calculated by co- donW (J. Peden, University of Nottingham; http://molbiol.ox.ac.uk/cu/). Intrageno- mic sequence similarity searches were performed by the graphical dotplot program Dotter (22).

Example 5. Vaccine efficacy. In order to study the efficacy of vaccine the following working scheme can for example be used:

1. Designing of PCR primers towards the VSP (variable surface protein) genes in a manner that the deduced amino acid sequence of the PCR products neither includes signal peptide (SP) motifs nor transmembrane helix (TMH) motifs. The primers both have so called handles in the 5 '-end. The upper primer handle has a restriction site for Notl and a 3C protease cleavage site. The lower primer handle is bi- otinylated in the 5 '-end, has a restriction site for Ascl and has an in- frame stop codon 3' ofthe ^scl site.

2. Designing of oligonucleotides for mutagenesis to substitute UGA codons, the universal stop codon that is coding for tryptophan in mycoplasmas, with UGG.

3. Amplification of the genes by PCR using the designed PCR primers.

4. Solid-phase restriction cleavage. The PCR product is immobilized onto strepta- vidin coated superparamagnetic beads by streptavidin-biotin interaction. Notl and suitable buffers are added to introduce a Notl overhang. The DΝA-bead complex is captured by a magnet and Ascl and suitable buffers are added after a couple of washes to separate the PCR product from the beads and to introduce an Ascl overhang. The beads are removed from the solution by magnet separation.

5. Cloning of the PCR products using pAffδc expression vector (Larsson et al, 2000) containing a His₆-tag, which is fused to the protein in the protein expression step. 6. DNA sequencing of the clones (colonies) to verify that they contain the correct plasmid insert.

7. Site-directed mutagenesis to substitute UGA codons with UGG.

8. Expression and purification of the fusion proteins, which here after are called re- combinantVSPs (rVSP), by immobilized metal ion affinity chromatography (IMAC).

9. Study of the immunoreactivity of the rVSPs with antibodies in blood sera of several cows infected by different strains of MmymySC.

10. Immunization of rabbits or other small laboratory animals with the rVSPs assisted with a suitable adjuvant to analyse the capacity of the rVSPs to induce immune response by measuring the amounts of IgG with enzymelinked immunosor- bant assay (ELISA).

11. Immunization of cattle with the rVSPs assisted with a suitable adjuvant or with placebo, and thereafter challenging them with a wide range of different MmymySC strains or with placebo.

12a. Serum is taken from each animal before vaccination, at challenge and at various occasions after the challenge (e.g. at day 7, 14 and 21) and is tested for IgG antibodies to MmymySC using ELISA.

12b. Clinical assessments are made by analysing the rectal temperature, respiratory signs, nasal discharge, severity and duration of cough, and arthritic lesions. 12c. Pathological post mortem examinations, where analyses of the severity of the gross pathological lesions of the inner organs are made.

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Claims

1. Variable protein of Mycoplasma mycoides subsp. mycoides biotype small colony chosen from:

(a) proteins chosen from the group comprising SEQ ID No 22-23 and 26-42.

(c) analogues or recombinant variants showing at least 50 %, preferably at least 70 %, more preferably at least 90 %, and most preferably at least 98 % ho- mology to the proteins of (a).

2. Variable protein of Mycoplasma mycoides subsp. mycoides biotype small colony chosen from:

(a) proteins chosen from the group comprising SEQ ID No 22-23 and 25-42.

(d) fragments having at least 20 consecutive amino acids in common with the proteins of (a); or

(e) analogues or recombinant variants showing at least 70 %, preferably at least 90 %, and more preferably at least 98 % homology to the proteins of (a); for use in a vaccine.

3. Nucleic acid molecule encoding a variable protein of claim 1.

4. Nucleic acid molecule of claim 3, chosen from the group comprising SEQ ID No 1-2 and 5-21.

5. Recombinant vector comprising a nucleic acid molecule of claim 3 or 4.

6. Fusion protein comprising a variable protein of claim 1.

7. Fusion protein comprising at least two variable proteins of claim 1 or 2, and at least one linker molecule positioned between the proteins.

8. Host cell comprising the recombinant vector of claim 5.

9. Vaccine composition comprising at least one protein according to claim 1-2, preferably a mixture of more than one protein, and optionally additional components.

10. Vaccine composition according to claim 9, wherein at least one protein comprises at least 60 amino acids, preferably at least 80 amino acids, more preferably at least 100 amino acids, most preferably at least 150 amino acids.

11. Vaccine composition according to any one of claims 9-10, for use in preventing disease caused by Mycoplasma capricolum, gallisepticum, genitalium, hominis, hyopneumoniae, mycoides, pneumoniae, specifically Mycoplasma mycoides.

12. Use of at least one protein according to claim 1-2 for the preparation of a vaccine composition for use in preventing disease caused by Mycoplasma capricolum, gallisepticum, genitalium, hominis, hyopneumoniae, mycoides, pneumoniae, specifically Mycoplasma mycoides.

13. Affinity reagents, such as antibodies and affibodies, that specifically binds to at least one protein according to claim 1.

14. Use of at least one protein according to claim 1 for diagnosing the occurrence of Mycoplasma capricolum, gallisepticum, genitalium, hominis, hyopneumoniae, mycoides, pneumoniae, specifically Mycoplasma mycoides.

15. A process for preparing a protein according to claim 1 comprising the steps of a) introducing a vector according to claim 5 into a suitable host cell; b) culturing the host cell under conditions that allows the expression of a protein according to claim 1; and c) isolating the protein of step b) from the host cell or cell culture supernatant.