FR2883978A1 - USE OF PROTEIN 2E9 IN IN VITRO DIAGNOSIS OF MYCOPLASMA PNEUMONIAE INFECTION - Google Patents

Abstract

The invention relates to the use of the protein 2E9, polypeptide sequence ID SEQ No. 2, or parts or variants of said protein, the use of sequences coding for said proteins and the use of antibodies directed against said proteins in the field of in vitro diagnosis of Mycoplasma pneumoniae infection.

Description

The present invention relates to the use of polypeptides and

  polynucleotides in the field of diagnosis of infection with the bacterium Mycoplasma pneumoniae.

  M. pneumoniae causes acute respiratory infections, more common in children from 5 years of age and in young adults. It can be atypical pneumonia, favorable evolution, sometimes associated with other evocative manifestations (ENT, cutaneous, hematological, neurological), or more often simple tracheobronchites. Its possible role in asthma is still a hypothesis.

  M. pneumoniae is thought to be responsible for about 15 to 20% of community-acquired pneumonia that is endemic with small outbreaks every four to seven years. The persistence of mycoplasma in the respiratory tract (between a few weeks and a few months after the start of infection) contributes to the endemic nature of the disease (Bébéar C, Bébéar CM and Barbeyrac B. in Freney J, Renaud F, Hansen W, Bollet C, Précis of Clinical Bacteriology, ESKA, Paris, 2000).

  M. pneumoniae enters the body by air (inhalation of droplets, direct contact with infected individuals) and adheres to respiratory epithelial cells. The bacteria produce peroxides that affect ciliary movement and produce cell damage and a local inflammatory response. Immune-pathological reactions cause the appearance of infiltrates and sometimes autoantibodies, due to the presence of M. pneumoniae-like membrane antigens identical to certain antigens found in the brain and pancreas (Waites Ken B, Talkington Deborah F, Mycoplasma pneumoniae and its role as a human pathogen, Clin Microbiol, Rev., 2004).

  Respiratory symptomatology does not differentiate M. pneumoniae infections from those caused by other atypical pneumonia agents. The onset of the disease is progressive after a fairly long incubation period of 15 to 20 days. The disease is manifested by fever, malaise, headache, myalgia and back pain and, most importantly, a dry, stubborn cough. The general state is little altered. Physical examination is poor in symptoms and contrasts with the importance of radiological images. The evolution is generally slowly regressive but the convalescence is long and the persistent cough. Complications and extra-pulmonary localization are possible: pleurisy, rash, sinusitis, myocarditis, pericarditis, joint involvement, haemolytic anemia, nerve manifestations, genital infections (Waites Ken B, Talkington Deborah F, Mycoplasma pneumoniae and its role as a human pathogen , Clin Microbiol, Rev., 2004, Bébéar C., Bébéar CM and Barbeyrac B. in Freney J., Renaud F., Hansen W., Bollet C., Précis de bacteriologie clinique, ESKA, Paris, 2000 ).

  The absence of cell wall in M. pneumoniae makes the bacterium insensitive to penicillin and other beta-lactams usually given in bacterial pneumonia. This natural resistance makes the diagnosis of M. pneumoniae infection essential for the proper administration of antibiotic therapy to the patient. Potentially active antibiotics are tetracyclines, fluoroquinolones, macrolides, and the like, with treatment usually based on macrolide administration.

  There is no standard diagnostic test for the detection of M. pneumoniae. The tests currently used are Culture and Polymerase Chain Reaction (PCR) for direct diagnosis and serological tests (cold agglutinins, Complement Fixation Reaction, indirect immunofluorescence, passive agglutination, ELISA), for indirect diagnosis.

  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 100% specific. The persistence of the bacteria can be up to several weeks after infection, and additional tests, such as the determination of specific antibodies, are needed to confirm the diagnosis of 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). Different systems have been proposed, amplification of random sequences, amplification of the adhesin gene or the gene coding for 16S RNA. This approach is sensitive (78-92%), allowing detection of 10 to 100 colony-forming units (cfu) with good specificity (92-100%) (Loens K, Goossens H and Ieven M., Molecular Diagnosis of Mycoplasma pneumoniae respiratory tract 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, as no detection kit is commercialized.

  Serologies are the most used methods in the diagnosis of M. pneumoniae infection, especially in the absence of samples. 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 and therefore it is recommended to search, in parallel, an IgG and IgM response. IgA is produced early in the course of infection but its level of production decreases more rapidly than that of IgG and IgM. Yet absent from existing serological tests, 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 GE Kenny. of mycoplasmal infections, Cumitech of the American Society for Microbiology, 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 can be implemented by means of a commercially available 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 (Waites KB, Babyar CM, Robertson JA, Talkington DF, and GE Kenny. of mycoplasmal infections.

  Cumitech of the American Society for Microbiology, coordinating editor: F. S. Nolte, 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, highly suggestive in children and adolescents, is more rarely observed in adults. It is preferable to look for specific antibodies on two sera taken 10-15 days apart to demonstrate seroconversion (x4 increase in antibody titre).

  In summary, current practices still do not meet medical expectations for diagnosing M. pneumoniae infections. Although serologic testing appears to be the most appropriate, the diagnosis of active infection remains, in many cases, difficult to differentiate from that of past infection (Waites K.B., C.M. Bébéar, J. A. Robertson, D. F. Talkington, and G.E.

  Kenny. Laboratory diagnosis of mycoplasmal infections. Cumitech of American Society for Microbiology, coordinating editor: F. S. Nolte., 2001, ASM press: 1-30, Talkington DF, Shott S, Fallon MT, Schwartz SB and Thacker WL. Analysis of eight commercial enzyme immunoassay tests for antibodies to Mycoplasma pneumoniae in human serum, Clin. Diagn. Lab. Immunol., 2004).

  The inventors of the present invention have identified, from the genome of M. pneumoniae, a gene coding for a protein that they called 2E9, of SEQ ID N 2 polypeptide sequence. The protein called 2E9 is a fragment of a protein homologous to a cytoplasmic protease involved in the stress response and found in other microorganisms.

  The inventors of the present invention have thus identified polynucleotides and polypeptides whose possible uses are described below.

  Definitions The following definitions are given to facilitate the understanding of certain terms used in this specification.

  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, an RNA composed of a mixture of one or more single-stranded region (s) and one or more regions ( s) double stranded 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, DNAs or RNAs, having skeletons modified for stability, or other reasons, are included in the term polynucleotides. 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 points of the polypeptide and anywhere in the polypeptide: in the peptide backbone, in the amino acid chain, or at the carboxy or aminoterminal ends.

  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. (PROTEINS STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., Creighton TE, WH Freeman and Company, New York (1993) and Wold, 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 182: 626-646 (1990) and Rattan et al., Protein Synthesis: Posttranslational Modifications and Aging, Ann NY Acad. Sci 663: 48-62 (1992)). 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 D. 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 polypeptide having, for example, an identity of at least 95% with the polypeptide ID SEQ N 2 is a polypeptide comprising at most 5 amino acids modified per 100 amino acids, with respect to said sequence. In other words, up to 5% of the amino acids in the sequence SEQ ID N 2 can be deleted or substituted by another amino acid or the sequence can comprise up to 5% more amino acids compared to the number total amino acid sequence ID SEQ N 2. These alterations of the sequence may be located at the amino and / or carboxyterminal 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 Genome 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, Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987).

  By analogy, a first polynucleotide having 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 said second polynucleotide may be deleted or substituted by another nucleotide, or said first polynucleotide may comprise up to 5% more nucleotides than the total number of nucleotides of the second polynucleotide. . These modifications may be positioned at the 3 'and / or 5' ends, or at any point between these ends, in one or more locations.

  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.

  By "culture medium" is meant the medium in which the polypeptide of 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 that of the polynucleotide according to the invention is to code 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.

  "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 ) (BIOCHEMISTRY, 4th Ed., L. Stryer, New York, 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 initial polynucleotide or the original polypeptide by one or more substitution (s), addition (s), deletion (s), fusion (s) or truncation (s) or more of these modifications. , taken in combination. A nonnatural 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.

  The subject of the invention is the use, in vitro, of the 2E9 protein, a portion or variants of this protein, of host cells comprising vectors including a polynucleotide encoding the 2E9 protein or a variant of the 2E9 protein. , in the production of antibodies and the field of in vitro diagnosis of Mycoplasma pneumoniae. The invention also relates to a diagnostic kit and a pharmaceutical composition. The subject of the present invention is the use, in the production of antibodies and in the field of the in vitro diagnosis of Mycoplasma pneumoniae infections, of a polypeptide comprising the amino acid sequence ID SEQ N 2 ( designated protein 2E9), encoded by the polynucleotide sequence ID SEQ N 1. The present invention also provides the use of a polypeptide comprising: a) a part of the amino acid sequence ID SEQ N 2 having the same function as the sequence ID SEQ N 2, or b) an amino acid sequence having at least 60% identity, preferably at least 80% identity, and more preferably at least 90% identity, with the acid sequence amino SEQ ID N 2, or with the portion of sequence defined under a), and having the same function as the sequence ID SEQ N 2.

  Thus, polypeptides according to the invention may comprise variants of the amino acid sequence ID SEQ N 2.

  In vitro diagnosis of Mycoplasma pneumoniae infection is carried out, for example, by standard immunoassay tests, such as ELISA or Western Blot. These tests use one of the polypeptides according to the invention which specifically binds to the serum antibodies against Mycoplasma pneumoniae present in the biological samples.

  The present invention also relates to the use of a polypeptide prepared by culturing a host cell comprising a recombinant vector having, inserted, a polynucleotide encoding said polypeptide.

  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, 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 the expression of the polynucleotide as well as polynucleotide sequences allowing the expression and transcription of a polynucleotide according to the invention and the translation of a polypeptide according to the invention. these sequences being chosen as a function of the host cells used.

  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; Streptomyces cells; insect cells, such as Drosophilia S2 cells and Spodoptera Sf9 cells; animal cells, such as CHO, COS, HeLa, C127, BHK, HEK 293 cells or plant cells.

  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 chromatographies.

  The subject of the present invention is also the use, in the production of antibodies and in the diagnosis of a Mycoplasma pneumoniae infection, of a polynucleotide comprising the polynucleotide sequence ID SEQ N 1 (referred to as 2E9 and extracted at from the genome of M. pneumoniae), encoding the protein 2E9. The invention also relates to the use of a polynucleotide comprising: a) a part of the sequence SEQ ID N 1 having the same function as the sequence ID SEQ N 1, or b) a polynucleotide sequence having at least 60% of identity, preferably at least 80% identity, and better still at least 90% identity, with the polynucleotide sequence ID SEQ N 1, or with the sequence part as defined under a), and having the same function as the sequence SEQ ID N 1, or c) a polynucleotide sequence complementary to the polynucleotide sequence ID SEQ N 1 or the part of sequence defined under a) or the sequence defined under b).

  The polynucleotides of the invention can be obtained by standard methods of DNA or RNA synthesis.

  The polynucleotides according to the invention may also comprise polynucleotide sequences such as 5 'and / or 3' noncoding sequences, such as, for example, transcribed sequences, non-translated sequences, splice signal sequences, sequences polyadenylated, binding sequences with ribosomes or sequences that stabilize the mRNA.

  Uses of the Antibodies and Polypeptides According to the Invention The present invention also relates to the use of a polypeptide according to the invention for detecting, in vitro, in biological samples the presence of antibodies directed against M. pneumoniae.

  The invention also relates to the use of antibodies according to the invention for detecting, in vitro, in biological samples the presence of M. pneumoniae antigens.

  The invention is further directed to the use of polypeptides and antibodies according to the invention to detect, periodically, in vitro, respectively, the antibodies directed against M. pneumoniae and the M. pneumoniae antigens and thus to follow the evolution of the pathology and the effect of a treatment applied to a patient.

  The biological samples tested may be blood, urine, saliva, serological puncture fluid (eg cerebrospinal fluid, pleural fluid or joint fluid) or one of their constituents (eg serum). Immunospecific antibodies can be obtained by administering a polypeptide 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.

  Kits The invention also relates to in vitro diagnostic kits comprising at least one of the polypeptides according to the invention, as well as in vitro diagnostic kits comprising at least one of the antibodies according to the invention. .

  Vaccines The present invention also relates to a pharmaceutical composition, usable as a vaccine, containing as active principle at least one polypeptide according to the invention or a polynucleotide or a recombinant vector or a host cell according to the invention.

  Experimental part A) Protocol for obtaining antigens Cloning of the coding sequence for the 2E9 protein The gene coding for the sequences of the 2E9 protein, which is an antigen, is obtained by PCR amplification from the genomic DNA of the M. pneumoniae bacterium (strain M129-B7, ATCC 29342) using as primer pair: E the sense oligonucleotide containing the sequence: 5'-CCAGCTGTAAAAAAACCAC -3 '; and E the antisense oligonucleotide containing the sequence: 5'-GTCCTTGCTTTCCTGCCACC -3 'In M. pneumoniae the TGA codon codes for a tryptophan whereas this same codon signals the translation stop in Escherichia coll. The underlined TGG codon in oligoantisens encodes a tryptophan in E. coli; this oligonucleotide thus allows the introduction of a silent mutation which replaces the wild-type TGA codon present in strain M129 (ATCC 29342). The production of the protein in E. coli is thus made possible without stopping the translation.

  The fragment thus amplified corresponding is cloned into a vector according to conventional techniques well known to those skilled in the art. This vector allows the production of the cloned protein under the control of an isopropyl-thiogalactoside inducible promoter (IPTG). The cloned protein corresponds to the amino acid sequence ID SEQ N 2.

  Expression of the Protein 2E9 An Escherichia coli strain is transformed by the expression vector previously described. The selected bacteria are grown overnight at 30 ° C. under agitation in 30 ml of Luria Bertani medium (LB, J. Miller, "A Short Course in Bacterial Genetics", Cold Spring Harbor Laboratory Press, 1992) containing ampicillin. at a final concentration of 100 μg / ml. The following day, the culture is diluted to 1 / 50th in a final volume of 1 liter of LB medium supplemented with ampicillin to a final concentration of 100 μg / ml and incubated at 30 ° C. with stirring. When the turbidity of the culture reaches an absorbance value at 600 nm (A600) of about 0.7, the production of the protein is induced by IPTG at a final concentration of 0.1 mM. The bacteria are harvested by centrifugation (10 minutes at 1400xg and at 4 C) when the turbidity of the culture reaches an A600 of about 1.5.

  Purification After centrifugation, the cells are resuspended in 20 mM Tris-HCl buffer at pH 8.0 containing 0.5 mM sucrose and then treated with lysozyme (0.2 g / l) in the presence of acid. 12.5 mM ethylenediaminotetraacetic acid (EDTA), DNase, RNase and PMSF (phenylmethylsulfonyl fluoride). The suspension is incubated for 4 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.

  After thawing, the bacteria are taken up in 25 mM Mes buffer at pH 6.0 and then sonicated 4 times in ice. After centrifugation at 15500xg at 4 ° C. for 30 minutes, the supernatant is filtered on 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 25 mM Mes [2-N-morpholinoethanesulfonic 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 at least overnight at 4 C and then centrifuged for 30 minutes at 20800xg. The pellet is then taken up in as small a volume as possible (in general 300 μl of 50 mM Na 2 HPO 4 / NaH 2 PO 4 buffer containing pH 8.0 containing 100 mM NaCl and then deposited on a column of gel filtration, for example Superdex HR 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 elecrophoresis analysis and mass spectrometry.

  B) In Vitro Diagnostic Assay Sera from patients who had a documented infection with M. pneumoniae (laboratory collection, ie, IgM titre or RFC titre positive) were used.

  The control sera corresponded to sera from blood donors (laboratory collection).

  The binding, on the purified recombinant 2E9 protein (obtained as described above), of the antibodies present in the sera was evaluated either by Western blotting tests or by the ELISA technique.

  Protocol of the Western blot test After transfer of the purified protein 2E9 onto a nitrocellulose membrane, the membrane is saturated for 30 minutes using a "phosphate buffered saline" (PBS) solution containing 3% of half milk. -skimmed. After washing three times with PBS containing 0.05% polyoxyethylene sorbitan (Tween), the membrane is placed in the presence of the serum tested at the appropriate dilution (ie 1 / 300th) in PBS buffer containing 3% semi-skimmed milk. for 45 minutes. After three further washes, alkaline phosphatase-labeled goat anti-human immunoglobulin G and / or A and / or M antibodies (e.g. 170-6462, Biorad) are added simultaneously or independently over a period of 30 minutes. after being diluted according to the supplier's protocol in PBS buffer containing 3% of half-milk. Three new washes are carried out and then 5-bromo4-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 a precipitation of the substrate on the membrane at the 2E9 position.

  Protocol of the ELISA Test The ELISA plates are left overnight at 4 ° C. in the presence of 0.5 μg of purified antigen (recombinant protein 2E9) in "phosphate buffered saline" (PBS). After four washes with PBS containing 0.05% polyoxyethylene sorbitan (Tween), the plates are saturated for 1 hour at 37 ° C. with PBS-Tween containing 5% semi-skimmed milk (250 μl per well). Four new washings are performed, then 100 μl of each positive serum, at the 1: 20 dilution in PBS-Tween buffer containing 5% of semi-skimmed milk, are added to each well. The plate is then left at 25 ° C. for 30 minutes. After four further washes, alkaline phosphatase-labeled goat anti-human immunoglobulin G and / or A and / or M antibodies are added (simultaneously or independently) for 30 minutes at 25 C after being diluted according to the supplier's protocol in PBS-Tween buffer containing 5% semi-skimmed milk. Four new washings are carried out and then 100 μl of substrate (p-nitrophenyl phosphate), pNPP, for example A-3469, Sigma) are added. The absorbance at 405 nm of each of the wells is measured after incubation for 30 minutes at 37 ° C.

  Results and Interpretation A standard result obtained is presented in the table below, knowing that the so-called "positive" sera in this table are those containing antibodies against M. pneumoniae identified by their binding with the polypeptides (antigens) of the invention. by ELISA: Table of results of the ELISA test Number of sera of patients infected with M. pneumoniae, diagnosed according to the prior art and subjected to the diagnosis according to the invention Serums "positive" in 16, ie 44% using antiretroviral agents. IgM as secondary antibodies among the 36 tested Blood donor sera 48 as controls Serum "positive" among the 1, or 20% tested. Significant results can be obtained using anti-IgA antibodies as secondary antibodies.

  The test is also applicable with anti-IgG as a secondary antibody.

  Similarly, similar results can be obtained by Western Blot.

  According to the Table, it is found that, by the ELISA test or by the Western blot test, at least 44% of M. pneumoniae infections are identified in vitro, thanks to the antigen according to the invention. 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) with respect to the protein 2E9 during M. pneumoniae infections and, secondly, that the 2E9 protein is relevant for the serological diagnosis of this type of infection.

Claims (9)

  1. Use for detecting, in vitro, the presence of antibodies to Mycoplasma pneumoniae in biological samples: a) amino acid sequence polypeptide ID SEQ N 2, or b) a portion of the acid sequence amino SEQ ID N 2 having the same function as the sequence ID SEQ N 2, or c) an amino acid sequence having at least 60% identity with the amino acid sequence ID SEQ N 2 or with the part of sequence as defined under b), and having the same function as the amino acid sequence ID SEQ N 2.   2. Use according to claim 1, characterized in that the polypeptide is prepared by culturing a host cell comprising a recombinant vector having inserted a polynucleotide encoding said polypeptide and by isolating said polypeptide from the culture medium.   3. Use according to claim 2, when it depends on claim 1 point a), characterized in that the polynucleotide is of sequence ID SEQ N 1.   4. Use for detecting, in vitro, the presence of antibodies to Mycoplasma pneumoniae in biological samples: a) of the polynucleotide of sequence ID SEQ N 1, or b) of a part of the sequence ID SEQ N 1 having the same function as the sequence ID SEQ N 1, or c) of a polynucleotide sequence having at least 60% identity with the polynucleotide sequence ID SEQ N 1 or with the sequence part as defined under b), and having the same function as the sequence ID SEQ N 1, or d) of a polynucleotide sequence complementary to a polynucleotide sequence as defined under a), b) or c).   5. Use of antibodies to detect, in vitro, the presence of Mycoplasma pneumoniae antigens in biological samples, characterized in that the antibodies are immunospecific for a polypeptide as defined in claim 1 or 2.   6. Kit for use according to any one of claims 1 to 4, characterized in that it comprises at least one of said polypeptides or one of said polynucleotides encoding said polypeptides.   7. Kit for use according to claim 5, characterized in that it comprises at least one of said antibodies.   8. Pharmaceutical composition, especially a vaccine, comprising as active principle at least: a) an amino acid sequence polypeptide ID SEQ N 2, or b) a part of the amino acid sequence ID SEQ N 2 having the same function as the sequence SEQ ID N 2, or c) an amino acid sequence having at least 60% identity with the amino acid sequence ID SEQ N 2 or with the portion of sequence as defined under b) , and having the same function as the amino acid sequence ID SEQ N 2 as well as a pharmaceutically acceptable excipient.   9. Pharmaceutical composition, in particular vaccine, comprising as active principle at least: a) a polynucleotide of sequence ID SEQ N 1, or b) a part of the sequence ID SEQ N 1 having the same function as the sequence ID SEQ N 1, or c) a polynucleotide sequence having at least 60% identity with the polynucleotide sequence ID SEQ N 1 or with the portion of sequence as defined under b), and having the same function as the sequence ID SEQ N 1 or d) a polynucleotide sequence complementary to a polynucleotide sequence as defined under a), b) or c) and a pharmaceutically acceptable excipient.