FR2937738A1 - USE OF ISOLATED POLYPEPTIDES HAVING ANTIGENIC PROPERTIES TO MYCOPLASMA PNEUMONIAE, AND CORRESPONDING POLYNUCLEOTIDES AND ANTIBODIES - Google Patents

Abstract

The subject of the invention is the use of a polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18 sequences to detect, in vitro, in biological samples, the presence of antibodies produced as a result of Mycoplasma pneumoniae infection, either to induce an immune response against Mycoplasma pneumoniae.

Description

The invention relates to the use of newly selected polypeptides and corresponding polynucleotides, on the one hand, in the field of the serological diagnosis of infections involving Mycoplasma pneumoniae - more precisely as a tool for the serological diagnosis of such infections - and, on the other hand, on the other hand, in the field of the prevention of infections by this bacterium. It also relates to the use of such polypeptides in the generation of antibodies suitable for detecting antigens of Mycoplasma pneumoniae. In all cases, it is the antigenic properties of said Mycoplasma pneumoniae polypeptides that are exploited, either to recognize the antibodies generated by the organism infected with Mycoplasma pneumoniae, or to cause said organism to generate such antibodies. for the purpose of immune protection or for the detection of Mycoplasma pneumoniae antigens. Mycoplasma pneumoniae is a wall-free bacterium that invades the cells of the respiratory epithelium. It is responsible for acute respiratory infections commonly found in children from the age of five and in young adults. It may be atypical pneumonia with favorable evolution, sometimes associated with other ENT, cutaneous, hematological, neurological or more often tracheobronchitis manifestations. Mycoplasma pneumoniae is said to be responsible for 15 to 20% of community-acquired pneumonia that is endemic with small outbreaks every four to seven years (Bébéar C, Bébéar CM and Barbeyrac B. in Freney J. Renaud F. Hansen W Bullet C. Precis of Clinical Bacteriology, ESKA, Paris, 2000). There is a first peak of incidence in children aged 5 to 15, in whom Mycoplasma pneumoniae is responsible for nearly 40% of community-acquired pneumonia and nearly 20% of the 35 community-acquired pneumonia requiring hospitalization. The second peak of incidence is in the adult after age 50, with numbers rising gradually with age to exceed 0% (Brunner H. Mycoplasma pneumoniae infections, Isr.J Med Sci (1981), 17). : 516-523, Ghosh et al., Surveillance of Mycoplasma pneumoniae infections in Scotland, 1986-1991, J Infect (1992), 25: 221-227, Waites et al., Mycoplasma pneumoniae and its vole as a human patgogen, Clin.Microbiol.Rev. (2004) 17: 697-728). The significant delay between primary infection in children and reinfection in adults shows that infection in children confers on the individual an effective immune protection against reinfection. A clinical association between M. pneumoniae and asthma in children has also been observed, although the nature of the correlation is not yet clearly established (Hansbro PM et al., Pole of atypical bacterial infection of the lung in predisposition / protection of asthma, Pharmacology and Therapeutics (2004), 101: 193-210). There appears to be a frequent underestimation of acute and even chronic infections with M. pneumoniae in children through conditions such as asthma or community-acquired pneumonia. Mycoplasma pneumoniae enters the body by air (inhalation of droplets, direct contact with infected subjects) by adhering to the cells of the respiratory epithelium. This contact generates oxidative stress at the origin of the alteration of the ciliary movement and the formation of cellular lesions. A local inflammatory reaction is formed. The immunological reaction can lead to the appearance of infiltrates and sometimes even autoantibodies. Some membrane antigens of M. pneumoniae are indeed similar to antigens found in the brain and pancreas (Waites KB, Talkington OF, Mycap / asmapneumoniaeand its rote as a human pathogen, Clin Microbiol Rev, 2004). Many infectious agents, whether viral or bacterial, can cause pneumonia, and the respiratory symptomatology makes it difficult to differentiate M. pneumoniae infections from those caused by other atypical pneumonia agents. . The onset of the disease is progressive after an incubation of 15 to 20 days. The disease is manifested by fever, malaise, headache, myalgia and rachialgia and especially a dry and obstinate cough. The general state is little altered and the physical examination poor in symptoms, contrasting with the importance of radiological images of the lungs. The disease is usually regressive over time; however, the convalescence is long and the cough persistent. M. pneumoniae can also cause various extrapulmonary complications by spreading to other organs: pleurisy, rash, sinusitis, myocarditis, pericarditis, joint involvement, haemolytic anemia, nerve manifestations, genital infections (Waites KB, Talkington OF, Mycoplasma pneumoniae and its rote as a human pathogen, Clin Microbiol Rev, 2004). The treatment of Mycoplasma pneumoniae infections is based on a probabilistic choice according to age, especially in children, and clinical and radiological criteria, neither of which is very specific or sensitive. It has been shown that the severity of the disease is often related to delayed antibiotic therapy. A study in 2007 reported the death of an 18-year-old girl in Austria after an infection with M. pneumoniae, as antibiotic therapy was probably not given early. The absence of a wall renders this bacterium insensitive to penicillin and other beta-lactams usually prescribed in case of pneumonia of bacterial origin. Thus the antibiotics frequently used in M. pneumoniae infections are tetracyclines, fluoroquinolones, macrolides and the like. However, cases of resistance to fluoroquinolones and macrolides have already been observed (Matsuoka, et al., Characterization and Molecular Analysis of Macrolide-Resistant Mycoplasma pneumoniae clinical isolates obtained in Japan, Antimicrob Agents Chemother, 2004, 48,12: 4824-4630; Gruson D. eta / In Vitra Development of Resistance and Six Fluoroquinolones in Mycoplasmspneumoniaeand Mycoplasmahominisrespectively Antimicrob Agents Chemother, 2005, 49, 3: 1190-1193). These resistance issues make the diagnosis of M. pneumoniae infection essential for antibiotic therapy to be appropriate and administered early.

At present, the direct diagnosis is made by culture or polymerase chain reaction (PCR); for the indirect diagnosis, serological tests exist such as cold agglutinins, complement fixation reaction, indirect immunofluorescence, passive agglutination and ELISA tests. However, there is no standard diagnostic test for the detection of M. pneumoniae infections. The culture of M. pneumoniae can be performed from throat swabs, nasopharyngeal aspirations in children and bronchoalveolar lavage, but it is long (2 to 3 weeks) and tedious. Rarely practiced in routine, it is rather reserved for the reference laboratories. However, when it is positive, it is specific to 100. The persistence of the bacterium can be up to several weeks after infection, additional tests, such as the determination of specific antibodies, are necessary to confirm the diagnosis of the infection. an active infection.

PCR gene amplification, based on samples taken from the respiratory tract, is a faster and more sensitive method than culture (out of 100 positive PCR samples, only 60 are positive in culture). Various systems have been proposed, such as the amplification of random sequences and the amplification of the adhesin gene or the gene encoding 16S RNA. This approach is sensitive (78-92%), allowing the detection of 10 to 100 cfu with a good specificity (92% to 100%) in K, Goossens N and Leven M., Molecular Diagnosis of Mycaplasma tiremaniaerespiratorytract infections, J. Clin. Microbiol., 2003). However, the technique is not standardized and remains very expensive and complex to use routinely in most clinical microbiology laboratories; no detection kit is marketed.

Serologies are the most widely used methods for the diagnosis of M. pneumoniae infection, particularly in cases of absence of specimens, such as nasopharyngeal aspirations or bronchoalveolar lavage. Following an infection with M. pneumoniae, the immune system of a non-immunosuppressed individual responds rapidly with antibody production reaching a peak after 3 to 6 weeks and then gradually declines over a period of a few months. to a few years. The production of M. pneumoniae-specific immunoglobulin M (IgM) occurs 7 to 10 days after the onset of infection. Their demonstration is often evidence of recent infection, especially in young children who have not had repeated exposure to the bacteria. However, in adults who have had repeated exposure, infection with M. pneumoniae does not cause a rapid rise in IgM and, in this case, marketed serological tests demonstrating an IgM response may be defective. Similarly, the IgM response may persist for months or years, the level of IgM antibody does not necessarily reflect a recent infection. In some cases, reinfection leads to an increase in the level of IgG, which is why it is recommended to look for an IgG and IgM response in parallel. IgA is produced early in the course of infection but its level of production decreases more rapidly than that of IgG and IgM. They may be good indicators of recent infection for all age groups, even after multiple re-infections (Waites KB, Babyar CM, Robertson JA, Talkington DF, and Kenny GE, Laboratory diagnosis of mycoplasmal infections.) Cumitech of American Society for Microbiology, co-authoring editor: FS Nolte., 2001, ASM press: 1-30). There are several, often complementary, serological techniques for diagnosing M. pneumoniae infection. The main tests are cold agglutinins, complement fixation, indirect immunofluorescence, passive agglutination tests and ELISA tests.

The presence of cold agglutinins, suggestive at a rate> 64, is sometimes found in M. pneumoniae infections but is neither constant nor characteristic. This previously used technique is therefore no longer recommended in the search for M. pneumoniae infection. The complement fixation reaction (RFC), using an antigenic preparation made from the entire microorganism, has long been used. The test measures the IgM and IgG levels simultaneously without differentiating them. A title> 64, is suggestive of an infection. The technique is cumbersome, insensitive and can lead to cross-reactions or uninterpretable results (anticomplementary sera). The search for specific antibodies can be done by indirect immunofluorescence. The serum to be tested, put in contact with M. pneumoniae antigens, is revealed by anti-IgM or anti-human IgG antibodies conjugated to a fluorochrome. This technique exists as a commercial kit but requires a fluorescence microscope. The reading of the slides is long and tedious and the interpretation of the results remains delicate.

Passive agglutination tests for the detection of IgM and / or IgG are commercially available. They demonstrate recognition by antibodies, contained in the patient's serum, antigens (extracts of M. pneumoniae) attached to latex particles, gelatin or even erythrocytes in the case of indirect haemagglutination test (IHA). The technique requires at least two sera to demonstrate an increase in antibody titer and has no advantages over the ELISA technique Maltes K.B., C.M. Bébéar, J. A. Robertson, O.F. Talkington, and G. E. Kenny. Laboratory diagnoses of mycoplasmal infections, Cumitech of the American Society for Microbiology, coordinating editor: F.S. Nuite, 2001, ASM press: 1-30). ELISA techniques can independently detect IgG or IgM. Preparations of bacterial extracts, purified proteins such as adhesin P1, glycolipids or synthetic peptides were used, fixed to the solid support. The patient serum is incubated with the antigenic solid phase, and anti-human IgG or anti-IgM antibodies, conjugated to an enzyme, react with the antigen-bound antibodies. The complex is evidenced by the hydrolysis of a substrate of the enzyme, resulting in a colored product. The choice of ELISA (IgM and / or IgG) depends on the age of the patient and the number of sera that can be tested. The presence of IgM is very suggestive in children and adolescents but more rarely seen in adults. It is preferable to look for specific antibodies on two sera taken 10-15 days apart, to demonstrate seroconversion (increase x 4 of the antibody titre). In summary, current practices still do not meet medical expectations to establish definitively the diagnosis of M. pneumoniae infections in children or adults. Although serological tests seem to be the most appropriate, in many cases the diagnosis of active infection remains difficult to distinguish from that of past infection (Waites KB, Babyar CM, JA Robertson, Talkington DF, and GE Kenny. Laboratory diagnosis of mycoplasmal infections, Cumitech of the American Society for Microbiology, coordinating editor: FS Halte, 2001, ASM press: 1-30, Talkington OF, Shott S, Fallon MT, SB Schwartz and Thacker WL. test for detection of antibodies to Mycop / asmapneumonissin human serum, Clin Diagn Lab Immunol., 2004). The genome of M. pneumoniae was sequenced in 1996 and re-annealed in 2000 (Himmelreich R., Hilbert H., Plagens H., Pirkl E., Li BC., Herrmann R. Complete sequence analysis of the gem of the Mycop bacteria / Nucleic Acid Research, Vol 22, No 24, 4420-4449, Dandekar T., Huynen M., Doerks T., Sanchez-Pulido L., Snel B., Suyama M., Yuan YP, Hermann R., Bork P. , Reannotating the Mycoplasmapneumonia genome sequence: adding value, function and reading frames.Nucleic acid research, Vol 28, No 17, 3278-3288.) It was possible to identify 688 open reading phases. Following selective investigations, the inventors of the present invention have identified, from the genome of M. pneumoniae, new polynucleotides and new polypeptides whose possible uses are described below. The inventors had to set up a selective screen to identify the unique antigenic properties of said polypeptides. The polypeptides have been specifically selected for their antigenic properties, among many others derived from M. pneumoniae, some of which are localized on the surface of the bacterium or intrinsic to the membrane and which have proved irrelevant to the serological diagnosis of such an infection. Although the DNA sequence of these polypeptides was identified during the sequencing of the genome, the state of the art did not in any way make it possible to demonstrate the antigenicity of said polypeptides.

The present invention aims to solve the deficiencies of current serological tests and to allow the diagnosis of M. pneumoniae in children or adult patients suffering from pneumonitis or asthma. It also proposes to provide pharmaceutical compositions for the prevention of M. pneumoniae infections.

Definitions The following definitions are given in order to facilitate the understanding of certain terms used in this description. The term "polynucleotide" is understood to mean a polyribonucleotide or a polydeoxyribonucleotide which may be a modified or unmodified DNA or RNA. The term polynucleotide includes, without limitation, single-stranded or double-stranded DNA, a DNA composed of a mixture of one or more single-stranded region (s) and one or more double-stranded region (s), a DNA which is a mixture of single-stranded, double-stranded and / or triple-stranded regions, single-stranded or double-stranded RNA, RNA composed of a mixture of one or more single-stranded region (s) and one or more double-stranded region (s) and hybrid molecules comprising DNA and RNA which may comprise single-stranded, double-stranded and / or triple-stranded regions or a mixture of single-stranded and double-stranded regions. The term polynucleotide may also include RNA and / or DNA comprising one or more triple-stranded regions. The strands in such regions can come from the same molecule or from different molecules. Therefore DNA or RNA, having skeletons modified for stability or other reasons, are included in the term polynucleotide. By polynucleotide is also meant DNAs and RNAs containing one or more modified bases. By "modified base" is meant, for example, unusual bases such as inosine. The term polynucleotide also refers to polynucleotides of chemically, enzymatically or metabolically modified form. Polynucleotides also include short polynucleotides such as oligonucleotides. The term "polypeptide" means a peptide, an oligopeptide, an oligomer or a protein comprising at least two amino acids joined to each other by a normal or modified peptide bond. The term polypeptide includes short chains, called peptides, oligopeptides and oligomers, and long chains, called proteins.

A polypeptide may be composed of amino acids other than the 20 amino acids encoded by human genes. A polypeptide may also be composed of amino acids modified by natural processes, such as the post-translational processing process or by chemical methods, which are well known to those skilled in the art. The same type of modification may be present at several sites of the polypeptide and anywhere in the polypeptide: in the peptide backbone, in the amino acid chain, or at the carboxy or amino terminus. A polypeptide may be branched following ubiquitination or cyclic with or without branching. This type of modification may be the result of natural or synthetic post-translation processes, which are well known to those skilled in the art. For example, by modification of a polypeptide is meant acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme, covalent attachment of a nucleotide or a nucleotide derivative, the covalent attachment of a lipid or a lipid derivative, the covalent attachment of a phosphatidylinositol, covalent or non-covalent crosslinking, cyclization, disulfide bond formation, demethylation , cysteine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodization, methylation, myristoylation, oxidation, process proteolytic, phosphorylation, prenylation, racemization, seneloylation, sulfation, amino acid addition, such as arginylation or ubiquitination. (2nd ed., TE Creighton, WN Freeman and Company, New York (1993) and Wald, F., Posttranslational Protein Modifications: Perspectives and Prospects, pp. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, BC Johnson, Ed., Academic Press, New York (1983), Seifter et al., Meth Enzymol 1862: 826-646 (1990) and Rattan et al., Protein Synthesis: Posttranslational Modifications and Aging, Ann NY Acad. Sci 663: 48-62 (1992)).

"Isolated" means, modified by the hand of man from the natural state, that is to say that the polynucleotide or the polypeptide present in nature has been modified or isolated from its natural environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism is not "isolated", but the same polynucleotide or polypeptide separated from coexisting materials in its natural state is "isolated". The term "percent identity" between two polynucleotide or polypeptide sequences is the percentage of identical nucleotides or amino acids between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences. being randomly distributed over their entire length. Comparisons between two polynucleotide or polypeptide sequences are traditionally performed by comparing these sequences after optimally aligning them, said comparison being made by segment or "comparison window" to identify and compare the local regions of sequence similarity. This comparison can be carried out by means of a program, for example the EMBOSS-Needle program (global alignment Needleman-Wunsch) using the matrix BLOSUM62 / Open Gap 10.0 and Extension Penalty of 0.5 (Needleman, SB and Wunsch, CD (1970), J. Mol Biol., 48, 443-453 and Kruskal, JB (1983), An overview of sequence comparison, In O. Sankoff and JB Kruskal, (ed), Time warps, strind edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley). The percentage of identity is calculated by determining the number of identical positions for which the nucleotide or amino acid is identical between the two sequences, by dividing this number of identical positions by the total number of positions in the comparison window and by multiplying the result obtained by 100. A first polypeptide having, for example, an identity of at least 95% with a second polypeptide is a polypeptide comprising, at most, 5 modified amino acids per 100 amino acids, with respect to the sequence of said second polypeptide. In other words, up to 5% of the amino acids of said second polypeptide may be deleted or substituted with another amino acid or said first polypeptide may comprise up to 5% more amino acids than the total number of amino acids. amino acids of the second polypeptide. These modifications of the sequence may be located at the amino and / or carboxy-terminal positions of the amino acid sequence or at any point between these terminal positions in one or more locations (Computational Molecular Biology, Lesk, AM, ed. , Oxford University Press, New York, 1988, Biocomputing: Informatics and Gemme Projects, Smith, DW, ed., Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994. Analysis in Molecular Biology sequence, von Heinje, G., Academic Press, 1987).

By analogy, a first polynucleotide having, for example, an identity of at least 95% with a second polynucleotide is therefore a polynucleotide having, at most, 5 modified nucleotides per 100 nucleotides, relative to the sequence of said second polynucleotide. In other words, up to 5% of the nucleotides of the polynucleotide, for example, of sequence ID SEQ No. 1 may be deleted or substituted by another nucleotide, or a polynucleotide may comprise up to 5% of nucleotides in addition. relative to the total number of nucleotides of the polynucleotide ID SEQ No. 1. These modifications may be positioned at the 3 'and / or 5' ends, or at any point between these ends, in one or more locations. "Polypeptide fragment" is understood to mean a polypeptide comprising at least "n" consecutive amino acids derived from the polypeptide ID SEQ No. 2, 4, 6, 8, 10, 12, 14, 16, 18 where, according to the sequences, n will be equal to 7 or more amino acids (e.g., 8, 10, 12, 14, 16, 18, 20, 30, 40, 50 or more amino acids). Preferably, said fragments comprise an epitope of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 sequences. These B or T type epitopes can be determined by software (for example, PEOPLE [Alix, 1999], PREDITOP [Pellequer and Westhof, 1993] or TEST [Zao et al., 2001]) or experimentally by standard techniques [eg, epitope mapping or mild proteolysis]. "Polynucleotide fragment" is understood to mean a polynucleotide comprising at least "n" consecutive nucleotides derived from polynucleotides ID SEQ No. 1, 3, 5, 7, 9, 11, 13, 15, 17 where, according to the sequences, n will be equal to 21 nucleotides or more (e.g., 22, 23, 24, 25, 30, 36, 42, 48, 54, 60, 90, 120, 150 or more nucleotides).

By "host cell" is meant a cell that has been transformed or transfected, or is capable of transformation or transfection, by an exogenous polynucleotide sequence. "Specific primers" are understood to mean short nucleotide sequences capable of hybridizing in a specific manner, thanks to the complementarity of the bases, on the DNA strand or on its complementary strand.

By "culture medium" is meant the medium in which a polypeptide according to the invention is purified. This medium may consist of the extracellular medium and / or the cell lysate. Techniques well known to those skilled in the art also enable the skilled person to restore active conformation to the polypeptide, if the conformation of said polypeptide has been altered during isolation or purification. By "function" is meant the biological activity of a polypeptide or polynucleotide. The function of a polypeptide according to the invention is that of a M. pneumoniae antigen, and the function of a polynucleotide according to the invention is that of coding this polypeptide. By "antigen" is meant any compound which, alone or in combination with an adjuvant or carrier, is capable of inducing a specific immune response. This definition also includes any compound having a structural analogy with said antigen capable of inducing an immunological response directed against said antigen. The function of an antigen of M. pneumoniae is thus to make it possible to establish the diagnosis of an infection with M. pneumoniae, but perhaps also to carry out therapeutic follow-ups or to specify if it is about a active or acute infection, for example, by the use of the detection of different isotypes of antibodies. The function of an M. pneumoniae antigen may also be to induce an immune response in a patient to whom it has been administered for the purpose of preventing subsequent infection with M. pneumoniae.

"Structural analogy" is understood to mean an analogy of the primary structure (sequence) as well as of the secondary structure (structural elements), of the tertiary structure (three-dimensional structure) or of the quaternary structure (association of several polypeptides in the same complex ) (BIDCHEMISTRY, 4th Ed., L. Stryer, New Ynrk, 1995).

The term "variant" of a so-called initial polynucleotide or of an initial said polypeptide, respectively, a polynucleotide or a polypeptide which differs by at least one nucleotide or an amino acid, but which retains the same intrinsic properties, that is, the same function. A difference in the polynucleotide sequence of the variant may or may not alter the amino acid sequence of the polypeptide it encodes, relative to an initial polypeptide. Nevertheless, by definition, these variants must confer the same function as the initial polynucleotide sequence, for example, encode a polypeptide having an antigenic function. The variant polynucleotide or polypeptide generally differs from the original polynucleotide or the original polypeptide by substitution, addition, deletion, fusion or truncation or a combination of these modifications. A non-natural variant of an initial polynucleotide or an initial polypeptide can be obtained, for example, by site-directed mutagenesis or by direct synthesis. A polynucleotide which can be hybridized with this polynucleotide sequence under stringent conditions is defined as "polynucleotide sequence complementary to the polynucleotide sequence".

The term "stringent conditions" is generally understood to mean, but not necessarily to, the chemical conditions that permit hybridization when the polynucleotide sequences have an identity of at least 80%. These conditions can be obtained according to methods well known to those skilled in the art. The term "antibodies" means monoclonal, polyclonal, chimeric, single chain, humanized antibodies, as well as Fab fragments, including the products of an Fab or of an immunoglobulin expression library.

An immunospecific antibody can be obtained by administering to an animal a given polypeptide, followed by recovering the antibodies produced by said animal by extraction from its body fluids. The animal may also be given a variant of said polypeptide, or host cells expressing this polypeptide.

The term "immunospecific" applied to the term antibody to a given polypeptide means that the antibody has a better affinity for that polypeptide than for other polypeptides known in the art.

By "affinity" is meant both a structural complementarity and a complementarity of low energy bonds between two molecules within the meaning of a commonly accepted definition (see, for example, Klotz 1M, Ligand-Protein Binding Affinities. Practical Approach (TE Creighton, Ed.), 1989, 25-35, IRL Press, Oxford, or Ajay, Murcko MA, Computational methods to predict binding free energy in ligand-receptor complexes, J. Med Cham 1995; : 4953-67). The affinity can be measured by conventional biochemical techniques (ELISA, competition, fluorescence, etc.) well known to those skilled in the art. By "positive" serum is meant a serum containing antibodies, produced as a result of infection with M. pneumoniae, identified by their binding to the polypeptide (antigen) of the invention. The term "sensitivity" refers to the proportion of infected patients diagnosed according to the prior art and given positive by the diagnosis according to the invention. "Specificity" is understood to mean the proportion of blood donors, tested as controls, subjected to the diagnosis according to the invention and given negative by the diagnosis according to the invention. By "diagnostic kit" is meant an assembly containing at least one of the products according to the invention (polypeptide, polynucleotide, antibody) and a suitable diluent, collected in a suitable container made of a suitable material. This container can gather the various complementary means necessary for the serological test (for example, labeled reagents, buffers, solutions which contain suitable ions, etc.) as well as the instructions required to carry out the test. The present invention meets the objectives it has set above by providing a novel polynucleotide and a novel polypeptide for the serological diagnosis of M. pneumoniae infections as well as a composition for the prevention of such infections. More specifically, the subject of the invention is the use, in vitro, of at least one polypeptide of sequence ID SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, or 18, a part or variants of these polypeptides, of host cells comprising vectors including a polynucleotide encoding at least one of said polypeptides or a variant of said polypeptides, in the production of antibodies and the field of in vitro diagnosis of L. pneumoniae. The invention also relates to a diagnostic kit and a pharmaceutical composition.

Use of polypeptides The identification of the polypeptides according to the invention is the result of a screen and in-depth studies that the sequences resulting from the genomic programs of M. pneumoniae did not suggest. The laboratory of the Depositor also knows polypeptides from M. pneumoniae that do not allow to diagnose an infection with this bacteria. The Applicant has, however, unexpectedly identified that such a diagnosis was possible using other polypeptides and even fragments of M. pneumoniae polypeptides that it specifically identified. Thus, the present invention relates to the use: or to detect, in vitro, in biological samples, the presence of antibodies produced following infection with Mycoplasma pneumoniae,. either to induce an immune response against Mycoplasma pneumoniae a) an isolated polypeptide of amino acid sequence ID SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18; or b) an isolated polypeptide consisting of a fragment of an amino acid sequence ID SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18 having the same function as said polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18; or c) an isolated polypeptide consisting of an amino acid sequence having at least 60% identity with ID SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18 or with a fragment of sequence identified under b), and having the same function as said polypeptide of sequence ID SEQ No. 2, 4, 6, 8, 10, 12, 14, 16 or 18, it being understood that "by the same function as said polypeptide "is an antigenic function of Mycoplasma pneumoniae. More specifically, the amino acid sequence SEQ ID NO: 2 (referred to as 10B2 protein) is encoded by the polynucleotide sequence ID SEQ ID No. 1 - the amino acid sequence ID SEQ ID No. 4 (called protein 10A2) is encoded by the polynucleotide sequence ID SEQ ID NO: 3 - the amino acid sequence ID SEQ ID No. 6 (referred to as 1OA1 protein) is encoded by the polynucleotide sequence ID SEQ ID NO: 5 - the amino acid sequence ID SEQ ID NO. 8 (referred to as 14G5 protein) is encoded by polynucleotide sequence ID SEQ ID NO: 7 - amino acid sequence ID SEQ ID NO: 10 (referred to as protein 15C6) is encoded by polynucleotide sequence ID SEQ ID NO: 9 amino acid ID SEQ ID NO: 12 (referred to as protein 15E6) is encoded by the polynucleotide sequence ID SEQ ID NO: 11; - The amino acid sequence ID SEQ No. 14 (called 14E5 protein) is encoded by the polynucleotide sequence ID SEQ No. 13; the amino acid sequence ID SEQ ID No. 16 (called protein 15D6) is encoded by the polynucleotide sequence ID SEQ No. 15; and - the amino acid sequence ID SEQ No. 18 (called 7D4 protein) is encoded by the polynucleotide sequence ID SEQ No. 17. Thus, polypeptides according to the invention may comprise amino acid sequences ID SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 or variants or fragments of said amino acid sequences . The present invention also relates to a method for preparing a polypeptide as defined above, wherein a previously defined host cell is cultured and said polypeptide is isolated from the culture medium. The polypeptide can be purified from the host cells, according to methods well known to those skilled in the art, such as precipitation with chaotropic agents such as salts, in particular ammonium sulfate, ethanol, acetone or trichloroacetic acid, or such means as acid extraction, ion exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography or exclusion chromatography.

The present invention also relates to the use of: to detect, in vitro, in biological samples, the presence of antibodies produced following infection with Mycoplasma pneumoniae,. either to induce an immune response against Mycoplasma pneumoniae a) an isolated polynucleotide of sequence of 10 nucleotides ID SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, or 17; or b) an isolated polynucleotide consisting of a fragment of a nucleotide sequence SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15 or 17 and having the same function as said sequence polynucleotide SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, or 17; or c) an isolated polynucleotide having at least 60% identity with a SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 or 17 nucleotide sequence or with a sequence fragment such as defined under b), and having the same function as said polynucleotide of sequence ID SEQ No. 1, 3, 5, 7, 9, 11, 13, 15 or 17; or d) a polynucleotide sequence complementary to the sequence SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15 or 17 or the polynucleotide according to points b) or c) above, 25 being understood that having the same function as said polynucleotide means a coding function of an antigenic polypeptide of M. pneumoniae SEQ sequence ID No. 2, 4, 6, 8, 10, 12, 14, 16 or 18. Thus polynucleotides according to the invention may comprise polynucleotide sequences ID SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 or variants or fragments of said sequences. The polynucleotides of the invention can be obtained by standard methods of DNA or RNA synthesis. The polynucleotides in accordance with the invention may also comprise polynucleotide sequences such as the 5 'and / or 3' non-coding sequences, such as, for example, transcribed sequences, untranslated sequences, splice signal sequences, polyadenylated sequences, binding sequences with ribosomes or sequences that stabilize the mRNA.

The subject of the present invention is also the use of the polypeptide according to the invention prepared by culturing a host cell comprising a recombinant vector having, inserted, a polynucleotide coding for said polypeptide according to the invention. the invention. Many expression systems can be used such as, for example, chromosomes, episomes, derived viruses. More particularly, the recombinant vectors used may be derived from bacterial plasmids, transposons, yeast episomes, insertion elements, chromosomal elements of yeasts, viruses such as baculoviruses, papillonna viruses such as SV40, vaccinia virus, adenovirus, fox pox virus, pseudorabies virus and retroviruses. These recombinant vectors can also be derivatives of cosmids or phagemids. The polynucleotide sequence may be inserted into the recombinant expression vector by methods well known to those skilled in the art. The recombinant vector may comprise polynucleotide sequences for controlling the regulation of polynucleotide expression as well as polynucleotide sequences allowing the expression and transcription of a polynucleotide of the invention and the translation of a polypeptide of the invention. these sequences being chosen as a function of the host cells used.

The present invention also relates to the use of a host cell comprising a recombinant vector according to the invention.

The introduction of the recombinant vector into a host cell can be carried out according to methods well known to those skilled in the art such as calcium phosphate transfection, cationic lipid transfection, electroporation, transduction or infection. The host cells may be, for example, bacterial cells such as streptococci, staphylococci, Escherichia coli or Bacillus subtilis cells, fungi cells such as yeast cells and Aspergillus cells, cells Streptomyces, insect cells such as Drosophilia S2 and Spodoptera Sf9 cells, animal cells, such as CHO, COS, HeLa, C127, BHK, HEK 293 cells or plant cells.

The invention also relates to the use of a polypeptide according to the invention for the generation of antibodies adapted to detect, in vitro, in biological samples, the presence of antigens resulting from a Mycoplasma pneumoniae infection. The invention is further directed to the use of such antibodies for periodically, in vitro, detection of M. pneumoniae antigens and thus to follow the evolution of the pathology and the effect of a treatment applied to a patient. . Immunospecific antibodies can be obtained by administering a polypeptide as defined according to the invention, a fragment thereof, an analogue or an epitope fragment or a cell expressing this polypeptide, to a mammal, preferably non-human, according to methods well known to those skilled in the art. For the preparation of monoclonal antibodies, standard methods of producing antibodies from cell lines such as the hybridoma technique, the trioma technique, the human B-cell hybridoma technique and EBV hybridomas. Antibody isotypes Immunoglobulins (or antibodies) consist of two different polypeptide chains: two light chains (L) of isotype [kappa] or [lambda], and two heavy chains (H) of isotype [gamma], [alpha], [mu], [delta] or [epsilon]. These four chains are covalently linked by disulfide bridges. Both types of H or L chains have regions that contribute to the attachment of antigens and are highly variable from one immunoglobulin to another. These regions determine the affinity of the antibodies for their ligand. The isotypes of the heavy chains make it possible to define the class of the immunoglobulin; there are 5 antibody isotypes (IgA, IgM, IgD, IgE and IgG). Subclasses, for example IgG1, IgG2, IgG3 and IgG4 are defined by sequence differences of the heavy chain. The different classes of immunoglobulins have their own physicochemical properties and their synthesis directly depends on the phases and levels of activation of the immune response. In humans, IgG is the main class of immunoglobulins: their serum concentration in adults ranges from 8 to 16 g / l. Their plasma half-life is about 3 weeks. IgA is the second class of serum immunoglobulins, after IgG, in terms of concentration (2 to 4 g / l). On the other hand, they represent the predominant class of immunoglobulins in secretions (respiratory, salivary, digestive secretions ..., milk, colostrum, tears). In terms of structure, IgA has the particularity to be in several molecular forms: in serum, the IgA can be in the form of monomers (predominant form) or in the form of dimers associated with a chain J (chain of junction ). The J chain is a cysteine-rich peptide of 137 amino acids (molecular weight: 15,000 Da) of plasmocytic origin; the IgAl subclass is predominant in the serum; in secretions, IgA, called secretory IgA, are in the form of dimers; they are therefore associated with a chain J, but they also include a secretory component. The IgA produced by the B cells is captured by the epithelial cells by a receptor (polyIgR) located at the basal pole of the cell. It is during the transfer of IgA through the epithelial cell, towards the apical pole of the cell, that the secretory component (molecular weight: 70,000 Da), or secretory part, is added. The role of the secretory patch is to protect secretory IgA from proteolytic enzymes present in secretions. Like IgA, IgM can be in two distinct molecular forms: a monomeric form: this is the form in which IgM is synthesized and inserted into the B cell membrane; a pentameric form: this is the form in which the IgMs are secreted. The basic monomers are linked by disulfide bridges. In addition, a J chain connects the end of 2 monomers. IgD represents less than 1% of serum immunoglobulins. They are usually coexpressed with IgM on the surface of B cells where they appear to act as a receptor for antigens. IgE is present in a normal subject only in trace amounts. Kinetics of antibody appearance and affinity The kinetics of appearance of specific antibodies and of a given isotype are still poorly understood. In general, the so-called naive B cells synthesize different IgMs which constitute a large repertoire of molecules capable of fixing antigens (Steven A. Frank., Immunology and Evolution of Infectious Disease. (2002), Princeton University Press, Princeton , USA). Thus, during the first exposure to an antigen, it will bind with low affinity to different IgM of the immune system. This interaction will nevertheless stimulate the division of the corresponding naive B cell. The division is even faster than the affinity of the IgM is strong which allows an initial selection of the best clones. Moreover, during the division, the genetic rearrangements allow the increase of the affinity of the antibodies. The constant immunoglobulin region also changes to produce IgG in the circulatory system and IgA on the mucosal surface (Steven and Frank 2002. Immunology and Evolution of Infectious Disease, Princeton University Press, Princeton, USA).

Uses of the Isotypes of Antibodies According to the Invention Those skilled in the art can therefore deduce from what is explained above that the presence of one or more classes of antibodies and the particular affinity of the antibodies have different implications from a medical diagnostic point of view in the differentiation of recurrent or non-recurrent infections, active, acute, chronic, latent, and / or recent infection. Serology The biological samples tested may be blood, urine, saliva, serological puncture fluid (e.g., cerebrospinal fluid, pleural fluid or joint fluid) or one of their constituents (eg for example, serum).

Vaccines The present invention also relates to a pharmaceutical composition, usable as a vaccine, containing as active principle at least one of the polypeptides, polynucleotides, recombinant vectors or host cells according to the invention, and a pharmaceutically acceptable excipient (for example, a aqueous sterile solution or not, which may contain an antioxidant or a buffer or a solute making the composition isotonic for body fluids).

Kits The invention also relates to in vitro diagnostic kits comprising, on the one hand: • at least one of the polypeptides according to the invention, or at least one of the polynucleotides encoding said polypeptides, or at least one of the antibodies according to the invention, and, on the other hand, at least one diluent (for example, a buffer, a saline solution, etc.).

Experimental part Following a Western blot, it was found that the proteins 10B2, 10A2, 10A1, 14G5, 1506, 15E6, 14E5, 15D6, 7D4 (SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18) have utility in the diagnosis of M. pneumoniae infections. Other proteins, such as the GroEL protein designated herein 2A9 (SEQ ID NO: 20), the DnaK protein named 6F2 (SEQ ID NO: 22) and the ClpB protein called 10E2 (SEQ ID NO: 24), which have also been tested, do not have the same antigenic properties as the polypeptides identified according to the invention. These proteins are encoded by sequenced genes, the sequence of which is known to all and accessible in the databases.

A - Protocol for obtaining antigens

A.1) Cloning of the coding sequence for the SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 polypeptides.

The genes coding for the sequences of the polypeptides ID SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 are amplified by PCR from the genomic DNA of the bacterium M pneumoniae (strain M129-B7, ATCC 29342) using, respectively, primers specific for SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23 sequences; these primers are chosen by those skilled in the art so as to frame the DNA sequence and amplify it specifically (for example, ID SEQ No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23). The corresponding fragments, thus amplified, are cloned into vectors according to standard techniques well known to those skilled in the art. These vectors allow the production of the cloned proteins under the control of an inducible promoter by isopropyl thiogalactoside (IPTG). The cloned proteins correspond to the polypeptides ID SEQ Nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24.

A.2) Protein Expression A strain of Escherichia coll. is transformed by the expression vector described above. The selected bacteria are cultured overnight at 30 ° C with stirring in 50 ml of Luria Bertani medium (LB, J.Miller, A Short Course in Bacteria Genetics, Cald Spring Harbor Laboratory Press, 1992) containing, respectively, ampicillin and chloramphenicol at a final concentration of 100 μg / ml. The next day, the culture is diluted 1: 50 in a final volume of 1 liter of LB medium to which ampicillin and chloramphenicol, respectively, have been added at a final concentration of 100 μg / ml, and incubated at room temperature. 30 ° C with stirring. When the turbidity of the culture reaches an absorbance value at 600 nm (abbreviated A600) of about 0.7, the production of the protein is induced by IPTG at a final concentration of 0.5 mM. The bacteria are harvested by centrifugation (6 minutes at 5240 rpm at 4 ° C) when the turbidity of the culture reaches an A600 of about 1.5.

A.3) Purification After centrifugation, the cells are resuspended in 20 mM Tris-HCl buffer, pH 8, containing 0.5 mM sucrose, and then treated with lysozyme (0.1 g / 50 ml). presence of 250 mM ethylenediaminetetraacetic acid (EDTA). The suspension is incubated for 30 minutes at 4 ° C. and then centrifuged for 10 minutes at 4 ° C. at 15500 × g. The pellet is frozen at -20 ° C for at least one night.

A.4) Example: Purification of 10A1 (SEQ ID NO: 6) The purification protocols differ for each protein according to its solubility and its specific physicochemical characteristics. We present, by way of example, the purification of the 10A1 protein. After thawing, the bacteria are taken up in 20 mM Mes buffer at pH 6.0, and then sonicated 4 times 20 seconds in ice. After centrifugation at 15500xg at 4 ° C for 30 minutes, the supernatant is filtered through a 0.22 μm porosity membrane. The filtrate is then deposited on a cation exchange column (for example, SP-Sepharose 12 ml, Amersham Biosciences). After washing the column, the protein is eluted with a linear gradient of 0 to 1 M NaCl in 20 mM Mes [2- [N-morpholino] ethanesulfonic acid] buffer pH 6.0 in 20 column volumes. Fractions containing the protein are pooled and the proteins precipitated with ammonium sulfate to a final concentration of 0.6 g / l. The solution is left overnight at 4 ° C and then centrifuged for 30 minutes at 20,800xg. The pellet is then taken up in as small a volume as possible (generally 300 μl) of 50 mM Na 2 HPO 4 / NaH 2 PO 4 buffer, pH 8.0, containing 100 mM NaCl, then deposited on a column of gel filtration, for example SuperdexHR 75. 10/30, Amersham).

The eluted fractions containing the protein are pooled and glycerol is added to a final concentration of 20%. The purified proteins are then stored at -20 ° C. until they are used in the tests.

The protein concentrations are determined spectrophotometrically from the absorption coefficients calculated by the Pace method (CN Pace, Vajdos F., Fee l., Grimsley G. and Gray T., (1995), Protein Science 4, 2411- 2423). Protein purity is verified by SDS-PAGE electrophoresis analysis and mass spectrometry.

B - In Vitro Diagnostic Assay To perform diagnostic tests, sera from patients, children and adults, who have had a documented infection with M. pneumoniae (i.e., IgM and / or IgG titres and / or or RFC and / or PCR positive and / or positive culture, laboratory collection) were used. The control sera correspond to sera from blood donors that did not have an infection with M. pneumoniae after test characterization (i.e. IgM and IgG and IgA titers in ELISA negative and RFC negative; of the laboratory). The binding, on the purified recombinant proteins (obtained as described above), of antibodies present in the sera was evaluated either by Western Blot tests or by the ELISA technique.

B.1) Western blot test protocol, for the polypeptides as defined according to the invention. After transferring purified recombinant proteins (obtained as described above) to a nitrocellulose membrane, the membrane is saturated for 45 minutes with a solution of phosphate buffered saline (PBS) containing 3% semi-skimmed milk. After three washes with PBS containing 0.05% polyoxyethylene sorbitan (Tween), the membrane is placed in the presence of the tested serum at the appropriate dilution (1/500) in PBS buffer containing 3% semi-skimmed milk. for 45 minutes. After three further washes, alkaline phosphatase-labeled goat anti-human immunoglobulin G and A and M antibodies (e.g. 170-1042, Biorad) are added over a period of 45 minutes after dilution according to the protocol. from the supplier, in PBS buffer containing 3% semi-skimmed milk. Three new washings are carried out and then 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium are added, according to the indications of the supplier, until the result is obtained. A positive result corresponds to the binding of the human anti-immunoglobulin antibody to a compound complex of the polypeptide according to the invention fixed to the membrane and of the human serum antibody specifically recognizing it, which results in a localized coloration at the level of of said complex.

B.2: Protocol of the ELISA test, for the polypeptides as defined according to the invention.

The ELISA plates are left overnight at 4 ° C. in the presence of 0.5 μg of purified antigen (proteins 10B2, 10A2, 10A1, 14G5, 15C6, 15E6, 14E5, 15D6, 7D4, 2A9, 6F2, 10E2) in phosphate buffered saline (PBS). After four washes with PBS containing 0.05% polyoxyethylene sorbitan (Tween), the plates are saturated for one and a half hours at 37 ° C with PBS-Tween containing 4% bovine serum albumin (BSA) (250). pl per well). Four new washings are carried out and then 100 μl of each positive or control serum are added, at the 1/100 dilution in 4% PBS-BSA buffer, to each well. The plate is then incubated at 37 ° C for 30 minutes. After four further washes, peroxidase-labeled goat anti-human immunoglobulin G and / or A and / or M antibodies (eg 31415, Pierce) are added (simultaneously and / or independently) in 30 minutes at 37 ° C. have been diluted, according to the supplier's protocol, in 4% PBS-BSA buffer. Four new washings are carried out and then 100 μl of tetrabenzimidine substrate (TMB) (for example HD979505, Pierce) are added per well. After incubation for about 15 minutes, 100 μl of sulfuric acid is added to each well. The absorbance at 450 nm of each well is then measured after 5 minutes. The serums identified by their binding with the polypeptides (antigens) as defined according to the invention are considered to be positive in ELISA.

C) Results and interpretations Typical results obtained are presented in the various examples below. The sera of patients (children and / or adults) considered positive are those containing antibodies against M. pneumoniae identified by ELISA, by their binding with the polypeptides (antigens) as defined according to the invention.

EXAMPLE 1 Results Obtained by Using the Polypeptides According to the Invention for the Detection of Specific Antibodies in Serum (ELISA) in Children The panel of samples tested consisted of sera from children patients whose infection with M. pneumoniae was diagnosed by the demonstration of an IgM and / or IgG positive response by ELISA, and / or the demonstration of of a positive titre after a test of complement fixation reaction and / or by the demonstration of the microorganism by culture of samples and / or amplification by PCR. The panel of control individuals consists of sera of children with pneumonitis, diagnosed negative for a M. pneumoniae infection according to the prior art. Tables 1 and 2 show the results obtained according to the invention for the polypeptides 10B2 (SEQ ID NO: 2), 10A2 (SEQ ID NO: 4), 10A1 (SEQ ID NO: 6), 14G5 (SEQ ID NO: 8). ), 15C6 (SEQ ID NO: 10), 15E6 (SEQ ID NO: 12), 14E5 (SEQ ID NO: 14), 15D6 (SEQ ID NO: 16), 7D4 (SEQ ID NO: 18), 2A9 ( SEQ ID NO: 20), 6F2 (SEQ ID NO: 22) and 10E2 (SEQ ID NO: 24) with secondary antibodies recognizing immunoglobulin G (Table 1) or M (Table 2) present in sera from sick patients or of individual witnesses.

Table 1: Results (ELISA) Obtained Using Anti-IgG as Secondary Antibodies Serums of Infected Children Serums of Children With Undiagnosed Pneumonopathies Diagnosed with M. pneumoniae According to the Prior Art, Tested as M. pneumoniae according to 1 prior art control Polypeptides Number Number of sera Number of Number of sera of "positive" according to "negative" sera according to tested sera tested the invention the invention tested 10B2 21 12 (57.1%) 20 20 (100.0%) ID SEQ # 2 10A1 20 6 (30.0%) 20 (100.0%) SEQ ID NO: 6 6 F2 SEQ ID NO: 22 20 1 (5.0%) 20 (100.0%) Table 2: Results (ELISA) obtained from using anti-IgM as secondary antibodies Serums of children with sera of children with pneumonia diagnosed with M. pneumoniae pneumoniae pneumoniae according to the art M. pneumoniae according to the prior art, tested as anterior witnesses Number of Number of sera Number of Polype serums of "positive" sera according to Number of tested "negatives" tested the invention sera tested according to the invention 10B2 21 (47.6%) 20 18 (90.0%) SEQ ID NO: 2 10A2 18 7 (33.3%) 18 18 ( 100.0%) SEQ ID NO: 4 14G5 21 9 (42.9%) 20 20 (100.0%) SEQ ID NO: 8 15C6 21 10 (47.6%) 20 19 (95.0%) SEQ ID NO: 10 15E6 21 11 (52.4%) SEQ ID NO: 12 14E5 21 10 (47.6%) 20 19 (95.0%) SEQ ID NO: 14 15D6 21 10 (47.6%) 20 19 (95.0%) SEQ ID NO: 16 7D4 21 8 (38.1%) 20 19 (95.0%) SEQ ID NO: 18 2A9 21 2 (9.5%) 20 19 (95.0%) SEQ ID NO: 20 6F2 20 1 (5.0%) 20 20 (100.0%) SEQ ID NO ° 22 10E2 20 3 (14.5%) 19 (95.0%) SEQ ID NO: 24 Similar results can be obtained by Western Blot.

EXAMPLE 2 Results Obtained by Using the Polypeptides According to the Invention for the Detection of Specific Antibodies in Serum (ELISA) in Adults

The panel of samples tested consisted of sera from adult patients whose infection with M. pneumoniae was diagnosed by the demonstration of an IgM and / or IgG positive response by ELISA, and / or the detection of of a positive titre after a test of complement fixation reaction and / or by the demonstration of the microorganism by culture of samples and / or amplification by PCR. The panel of control individuals consists of sera from adult blood donors. Tables 3 and 4 show the results obtained according to the invention for the polypeptides 10B2 (SEQ ID NO: 2), 14G5 (SEQ ID NO: 8), 15C6 (SEQ ID NO: 10), 7D4 (SEQ ID NO: 18 ), 6F2 (SEQ ID NO: 22) with secondary antibodies recognizing immunoglobulin G (Table 3) or M (Table 4) present in sera of sick patients or control individuals.

Table 3: Results (ELISA) Obtained Using Anti-IgG as Secondary Antibodies Serums of Adults with Serums of Adult Blood-Diagnosed Donorhoods Tested as M. pneumoniae by Prior Control Polypeptides Number of Serums Number of sera tested "positive" sera according to "negatives" according to the invention sera sera the invention tested 10B2 8 3 (37.5%) 29 (96.7%) SEQ ID NO: 2 6F2 SEQ ID NO: 22 8 0 (0.0%) 29 (96.7%) Table 4: Results (ELISA) obtained using anti-IgM as secondary antibodies Serums of adults with sera of adult donors pneumonia diagnosed with M. pneumoniae according to the art of blood Tested as anterior control Polypeptides Number of Number of sera Number Number of sera tested "positive" sera according to "negatives" according to the invention sera sera the invention tested 14G5 ID SEQ No. 8 8 3 (37.5%) 30 29 (96.7 %) 15C6 8 5 (62.5%) 30 30 (100.0%) ID SEQ ID NO: 10 7D4 8 4 (50.0%) 30 29 (96.7%) SEQ ID NO: 18 6F2 SEQ ID NO: 22 8 0 (0.0%) 29 (96.7%) Similar results can be obtained by Western Blot .5 From the results tables 1 to 4, it can be seen that, by the ELISA test, the proteins 2A9, 6F2 and 10E2 have poor performances (specificity and / or sensitivity) and can not be used in the context of the serologic diagnosis of M. pneumoniae infection in children or adults. On the other hand, it is possible to identify in vitro and with a specificity of 100%, up to 57.1% by detecting IgG and up to 52.4% by detecting IgM, pneumonia caused by M. pneumoniae in children through the use, according to the invention, of one or other of the polypeptides. Similarly, up to 37.5% and 62.5% of M. pneumoniae infections can be diagnosed in adult patients by detecting IgG and IgM respectively for 96.7% and 100% specificities. one or other of the polypeptides identified according to the invention. We thus find a significant level of antibodies directed against one or other of the polypeptides identified according to the invention in most of the children and adults tested. These antigens can therefore provide infected individuals with long-lasting immune protection against reinfection. They could therefore be used to prevent M. pneumoniae infections.

It is thus demonstrated, on the one hand, the existence, in humans, of a significant antibody response (the probability associated with an x2 test is less than 0.05) vis-à-vis the polypeptides identified according to the invention during M. pneumoniae infections and, on the other hand, that the 10B2, 10A2, 10A1, 14G5, 1506, 15E6, 14E5, 15D6 and 7D4 proteins are relevant for the serological diagnosis of this type of infection. , through pathologies such as pneumonia. polynucleotide sequences ID SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17

Claims (9)

REVENDICATIONS1. Use for detecting, in vitro, in biological samples, the presence of antibodies produced following infection with Mycoplasma pneumoniae, a) an isolated polypeptide of amino acid sequence ID SEQ ID NO: 2; or b) an isolated polypeptide consisting of a fragment of an amino acid sequence SEQ ID NO: 2, having the same function as said SEQ ID NO: 2 sequence polypeptide; or c) an isolated polypeptide consisting of an amino acid sequence having at least 60% identity with the SEQ ID NO: 2 sequence, or with a sequence fragment identified under b), and having the same function said polypeptide of sequence ID SEQ No. 2, it being understood that by "the same function as said polypeptide" is meant an antigenic function of Mycoplasma pneumoniae. 2. Use according to claim 1, characterized in that said polypeptide is prepared by culturing a host cell comprising a recombinant vector having inserted, a polynucleotide encoding said polypeptide and isolating said polypeptide from said culture medium. 25 3. Use according to claim 2, characterized in that said polynucleotide is of nucleotide sequence ID SEQ No. 1. 4. Use for detecting, in vitro, in biological samples, the presence of antibodies produced as a result of Mycoplasma pneumoniae infection, a) an isolated polynucleotide of nucleotide sequence ID SEQ ID NO: 1; or b) an isolated polynucleotide consisting of a fragment of a nucleotide sequence ID SEQ No. 1, and having the same function as said polynucleotide of sequence ID SEQ No. 1; orc) an isolated polynucleotide having at least 60% identity with a nucleotide sequence SEQ ID NO: 1 or with a sequence fragment as defined under b), and having the same function as said SEQ ID sequence polynucleotide No. 1; or d) a polynucleotide sequence complementary to the sequence SEQ ID NO: 1, or to the polynucleotide according to points b) or c) above, it being understood that by having the same function as said polynucleotide is meant a coding function of an M. pneumoniae antigenic polypeptide of SEQ ID No. 2 sequence. 5. Use of immunospecific antibodies of a polypeptide as defined in claims 1 to 3 for detecting, in vitro, in biological samples, the presence of antigens resulting from infection with Mycoplasma pneumoniae. 6. Kit for carrying out the use according to any one of claims 1 to 4, in the field of the detection, in vitro, in biological samples, of the presence of antibodies produced as a result of a Mycoplasma pneumoniae infection, characterized in that it comprises at least one of said polypeptides or one of said polynucleotides, as identified in any one of said claims 1 to 4, associated with at least one diluent. 7. Kit for carrying out the use according to claim 5, characterized in that it comprises at least one of said antibodies associated with at least one diluent. 8. Pharmaceutical composition, especially vaccine, for carrying out the use according to any one of claims 1 to 4, for inducing an immune response against Mycoplasma pneumoniae, comprising, as active principle, at least: a) an amino acid sequence polypeptide ID SEQ No. 2; orb) a polypeptide consisting of a fragment of an amino acid sequence ID SEQ No. 2 having the same function as said polypeptide of sequence ID SEQ No. 2; or c) a polypeptide consisting of an amino acid sequence having at least 60% identity with the SEQ ID NO: 2 sequence, or with a sequence fragment identified under b), and having the same function as said polypeptide of sequence ID SEQ No. 2 and a pharmaceutically acceptable excipient, it being understood that by having the same function as said polypeptide "is meant an antigenic function of M. pneumoniae. 9. A pharmaceutical composition, especially a vaccine, for carrying out the use according to any one of claims 1 to 4 for inducing an immune response against Mycoplasma pneumoniae comprising, as active principle, at least: a) d) a polynucleotide of nucleotide sequence ID SEQ No. 1; or b) a polynucleotide consisting of a fragment of a sequence of nucleotides ID SEQ No. 1 and having the same function as said polynucleotide of sequence ID SEQ No. 1; or c) a polynucleotide having at least 60% identity with a sequence of nucleotides ID SEQ No. 1 or with a sequence fragment as defined under b), and having the same function as said polynucleotide of sequence ID SEQ No. 1; or d) a polynucleotide sequence complementary to the sequence SEQ ID No. 1, or the polynucleotide according to points b) or c) above, and a pharmaceutically acceptable excipient, it being understood that by having the same function as said polynucleotide is understood to mean a coding function of an antigenic polypeptide of M. pneumoniae of sequence ID SEQ No. 2.