Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56911—Bacteria
  • G01N33/56933—Mycoplasma
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/0241—Mollicutes, e.g. Mycoplasma, Erysipelothrix
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/30—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
  • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
  • C07K16/1253—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/53—DNA (RNA) vaccination
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
  • G01N2333/30—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2469/00—Immunoassays for the detection of microorganisms
  • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

• 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 serological multiparametric diagnostic tool for such infections - and, of on the other hand, in the field of prevention of infections by this bacterium.
• 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 manifestations, cutaneous, hematological, neurological or more often tracheobronchitis.
• Mycoplasma pneumoniae is thought 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 , Bollet C, Précis of Clinical Bacteriology, ESKA, Paris, 2000).
• the second peak of incidence concerns the adult after 50 years, with numbers rising gradually with age to exceed 30% (Brunner H.
• 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 DF, Mycoplasma pneumoniae and its role as a human pathogen, Clin Microbiol Rev, 2004).
• M. pneumoniae infections can cause pneumonia, and the respiratory symptomatology makes it difficult to differentiate M. pneumoniae infections from those caused by other atypical pneumonia agents. .
• the beginning 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 DF, Mycoplasma pneumoniae and its role as a human pathogen, Clin Microbiol Rev, 2004).
• Mycoplasma pneumoniae infections 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.
• M. pneumoniae 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).
• M. pneumoniae infection 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.
• the immune system of a non-immunosuppressed individual responds rapidly with the production of antibodies reaching a peak after 3 to 6 weeks. gradually decline over a period of months to 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.
• the complement fixation reaction 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 heavy, not sensitive and may give rise to cross-reactions or uninterpretable results ("anticomplementary" sera).
• the search for specific antibodies can be done by indirect immunofluorescence.
• the serum to be tested, brought into 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.
• IgM and / or IgG 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, CM Babyar, JA Robertson, DF Talkington, and GE Kenny. diagnosis of mycoplasmal infections, Cumitech of the American Society for Microbiology, coordinating editor: FS 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's serum is incubated with the antigenic solid phase, and anti-IgG or anti-human IgG 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.
• polypeptides have been selected specifically for their antigenic properties, among many others from M. pneumoniae, some of which are localized on the surface of the bacterium or intrinsic to the membrane and which have proved irrelevant for 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 including the lack of specificity and allow the diagnosis of Mycoplasma infection, including M. pneumoniae in children or adult patients with pneumonia or asthma. It also proposes to provide pharmaceutical compositions, in particular for the prevention of Mycoplasma infections, in particular M. pneumoniae.
• polynucleotide is understood to mean a polyribonucleotide or a polydeoxyribonucleotide which may be a modified or unmodified DNA or RNA.
• 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.
• 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.
• polynucleotide is also meant DNAs and RNAs containing one or more modified bases.
• modified base is meant, for example, unusual bases such as inosine.
• polynucleotide also refers to polynucleotides of chemically, enzymatically or metabolically modified form. Polynucleotides also include short polynucleotides such as oligonucleotides.
• 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.
• 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 amino terminal 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.
• a polypeptide 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, the phosphorylation, prenylation, racemization, seneloylation, sulfation, addition of amino acids, such as arginylation or ubiquitination.
• polypeptide according to the invention comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, and 18, the homologous polypeptide thereof, or fragments thereof, as defined herein, may also include sequences useful for purification of proteins (purification tags), such as polyhistidine tags, and optionally a sequence for cleaving these tags, for example cleavage sites with proteases.
• purification tags such as polyhistidine tags
• a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, and 18, comprises 350, 400, 500 , or 1000 amino acids maximum.
• 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.
• 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”.
• 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 performed by segment or by "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, Time warps, strind edits and macromolecules : the theory and practice of sequence comparison, pp. 1-44 Addison Wesley).
• EMBOSS-Needle program global alignment Needleman-Wunsch
• the percentage of identity is calculated by determining the number of identical positions for which the nucleotide or the 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, in particular dividing by the total number of positions of the larger of the two aligned sequences, and multiplying the result 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 amino acids modified per 100 amino acids, relative to the sequence of said second polypeptide.
• 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.
• polypeptide homologous of a polypeptide comprising or consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, or 18, a polypeptide having at least 60% identity, preferably at least 80% identity, and more preferably at least 90% sequence identity with the polypeptide comprising or consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, or 18.
• 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.
• up to 5% of the nucleotides of the polynucleotide, for example, of sequence SEQ ID 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 SEQ ID 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 SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, wherein, 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).
• the "polypeptide fragment” according to the invention comprises at least 20 amino acids, more preferably at least 30 amino acids, and still more preferably at least 40 amino acids.
• the "polypeptide fragment” according to the invention comprises at most 100 amino acids, more preferably at most 80 amino acids, and still more preferably at most 60 amino acids.
• said fragments comprise an epitope of the sequences SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, or 18.
• T 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 conventional techniques [for example, by epitope mapping or proteolysis].
• Polynucleotide fragment is understood to mean a polynucleotide comprising at least "n" consecutive nucleotides derived from SEQ polynucleotides. ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 where, according to the sequences, n will be 21 nucleotides or more (e.g., 22, 23, 24, 25, 30, 36 , 42, 48, 54, 60, 90, 120, 150 or more nucleotides).
• 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.
• 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.
• 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.
• 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.
• 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.
• a homologous polypeptide of a polypeptide comprising or consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, or 18, or
• a fragment or part of a polypeptide comprising or consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, or 18, or the homologous polypeptide is said to have the same antigenic function or the same function of antigen, in particular Mycoplasma pneumoniae, the polypeptide comprising or consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, or 18, if it can be linked by minus an antibody directed against a polypeptide represented by a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, and 18.
• 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).
• 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".
• 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.
• 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.
• 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.
• affinity is meant both a structural complementarity and a complementarity of the low energy bonds between two molecules within the meaning of a commonly accepted definition (see, for example, Klotz IM, Ligand-Protein Binding Affinities. a Practical Approach (TE).
• serum a serum containing antibodies, produced following a Mycoplasma infection, in particular M. pneumoniae, identified by their binding with the polypeptide (antigen) of the invention.
• Stensitivity is understood to mean 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.
• test kit means 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 that it has set above by providing a new polynucleotide and a new polypeptide for the serological diagnosis of Mycoplasma infections, in particular M. pneumoniae, as well as a composition for the prevention of such infections. infections.
• the subject of the invention is the use, in vitro, of at least one polypeptide having the sequence 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 Mycoplasma, including
• the invention also relates to a diagnostic kit and a pharmaceutical composition.
• 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 Applicant's laboratory knows polypeptides derived from M. pneumoniae that do not allow the diagnosis of a 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.
• the subject of the present invention is the use:
• amino acid sequence SEQ ID NO: 6 (called 10A1 protein) is encoded by the polynucleotide sequence SEQ ID NO: 5;
• amino acid sequence SEQ ID NO: 8 (called the 14G5 protein) is encoded by the polynucleotide sequence SEQ ID NO: 7;
• amino acid sequence SEQ ID NO: 18 (called 7D4 protein) is encoded by the polynucleotide sequence SEQ ID NO: 17.
• polypeptides according to the invention may comprise amino acid sequences 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 process for preparing a polypeptide as defined above, in which 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, chromatography. hydroxylapatite or exclusion chromatography.
• the present invention also relates to the use of:
• polynucleotides according to the invention may comprise polynucleotide sequences SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 or variants or fragments of said sequences.
• 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 present invention also relates to the use of the polypeptide according to the invention prepared by culturing a host cell comprising a recombinant vector having inserted a polynucleotide encoding said polypeptide according to 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.
• 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.
• bacterial cells such as streptococci, staphylococci, Escherichia coli or Bacillus subtilis cells
• fungi cells such as yeast cells and Aspergillus cells
• cells Streptomyces 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 an infection with a bacterium of the genus Mycoplasma , especially by M. pneumoniae.
• the invention relates to the use of immunospecific antibodies of a polypeptide according to the invention for detecting, in vitro, in biological samples, the presence of antigens resulting from an infection with a bacterium of the genus Mycoplasma, especially by Mycoplasma pneumoniae.
• the invention is further directed to the use of such antibodies to detect, periodically, in vitro, the antigens of a bacterium of the Mycoplasma genus, in particular M. pneumoniae, 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.
• Immunoglobulins consist of two different polypeptide chains: two light chains (L) of isotypes [kappa] or
• 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.
• 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). From the structural point of view, IgAs have the particularity to present themselves in several molecular forms:
• the IgA in the serum, can be in the form of monomers (predominant form) or in the form of dimers associated with a J chain (connecting chain).
• the J chain is a cysteine-rich peptide of 137 amino acids (molecular weight: 15,000 Da) of plasmocytic origin; the IgAI subclass is predominant in the serum;
• IgA in secretions
• 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 lymphocytes is captured by the epithelial cells, by a receptor (polylgR) located at the basal pole of the cell. It is during the transfer of I 1 IgA across the epithelial cell, toward the apical pole of the cell, the secretory component (molecular weight 70,000 Da), or secretory piece is added.
• the role of the secretory patch is to protect secretory IgA from proteolytic enzymes present in secretions.
• IgM can be in 2 distinct molecular forms: a monomeric form: this is the form in which the IgMs are synthesized and inserted into the membrane of the B lymphocyte;
• a pentameric form this is the form in which the IgMs are secreted.
• the basic monomers are linked by disulfide bridges.
• a chain J 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.
• 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).
• serological puncture fluid eg cerebrospinal fluid, pleural fluid or joint fluid
• serum one of their constituents
• the present invention also relates to a pharmaceutical composition, usable as a vaccine, in particular for inducing an immune response against a bacterium of the genus Mycoplasma, in particular against Mycoplasma pneumoniae, containing as active principle at least one of the polypeptides, polynucleotides, recombinant vectors or cells according to the invention, and a pharmaceutically acceptable excipient (for example, a sterile aqueous solution or not, which may contain an antioxidant or a buffer or a solute making the composition isotonic for body fluids).
• a pharmaceutically acceptable excipient for example, a sterile aqueous solution or not, which may contain an antioxidant or a buffer or a solute making the composition isotonic for body fluids.
• the invention also relates to in vitro diagnostic kits comprising, on the one hand:
• At least one of the polypeptides in accordance with the invention or at least one of the polynucleotides encoding said polypeptides, or
• At least one of the antibodies in accordance with the invention that is to say in particular an immunospecific antibody of a polypeptide according to the invention, and, secondly, at least one diluent (for example, a buffer, saline solution ).
• diluent for example, a buffer, saline solution
• the present invention also relates to a method, especially in vitro, for determining whether an individual is infected with a bacterium of the genus Mycoplasma, in particular by Mycoplasma pneumoniae, comprising the detection of antibodies directed against at least one polypeptide represented by a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ
• SEQ ID NO: 6 SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 18, in a biological sample of the individual .
• SEQ ID NO: 2 SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO : 18, comprises bringing the biological sample into contact with:
• At least one polypeptide comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 18; or
• At least one homologous polypeptide comprising or consisting of a sequence sharing at least 60% identity with a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 18; or
• proteins 10B2, 10A2, 10A1, 14G5, 15C6, 15E6, 14E5, 15D6, 7D4 SEQ ID NO: 2, 4, 6,
• 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 are amplified by PCR from the genomic DNA of the bacteria M. pneumoniae (strain M129-B7, ATCC 29342) using, respectively, primers specific for sequences SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23; these primers are chosen by those skilled in the art so as to frame the DNA sequence and amplify it specifically (for example SEQ ID NO: 1, 3, 5, 7, 9, 1 1, 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).
• IPTG isopropyl thiogalactoside
• the proteins cloned correspond to the polypeptides SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24.
• A.2 Expression of Proteins
• An Escherichia coli strain is transformed by the previously described expression vector.
• 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", CoId Spring Harbor Laboratory Press, 1992) containing, respectively, ampicillin and chloramphenicol at a final concentration of 100 ⁇ g / ml.
• the next day, the culture is diluted 1 / 50th 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 ⁇ g / ml, and incubated at 30 ° C with stirring.
• the production of the protein is induced by I 1 I PTG at a final concentration of 0.5 m.
• 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.
• 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) in the presence of 250 ml. mM of ethylenediaminetetraacetic acid (EDTA).
• EDTA ethylenediaminetetraacetic acid
• the purification protocols differ for each protein according to its solubility and its own physicochemical characteristics.
• the purification of the 10A1 protein After thawing, the bacteria are taken up in 20 mM Mes buffer at pH 6.0, then sonicated 4 times 20 seconds in the ice. After centrifugation at 15500xg at 4 ° C. for 30 minutes, the supernatant is filtered on a 0.22 ⁇ m porosity membrane. The filtrate is then deposited on a cation exchange column (for example, SP-Sepharose 12 ml, Amersham Biosciences).
• a cation exchange column for example, SP-Sepharose 12 ml, Amersham Biosciences.
• 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 the smallest possible volume (generally 300 ⁇ l) of 50 mM Na 2 HPO 4 / NaH 2 PO 4 buffer, pH 8.0, containing 10 mM NaCl and then deposited on a column of gel.
• 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, 241 1 -2423). Protein purity is verified by SDS-PAGE electrophoresis analysis and mass spectrometry.
• control sera correspond to sera from blood donors who 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; laboratory).
• the binding, on the purified recombinant proteins (obtained as described above), of the antibodies present in the sera was evaluated either by Western blotting tests or by the ELISA technique.
• Example B.1 Western blot test protocol for polypeptides as defined according to the invention.
• the membrane After transfer to purified recombinant proteins (obtained as described above) on a nitrocellulose membrane, the membrane is saturated for 45 minutes with a solution of phosphate buffered saline (PBS) containing 3% of semi-skimmed milk. 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 (1/500) in PBS buffer containing 3% of semi-skimmed milk during 45 minutes. After three further washes, alkaline phosphatase-labeled goat anti-human immunoglobulin G and A and M antibodies (e.g.
• a "positive" result corresponds to the binding of the anti-human immunoglobulin antibody to a compound complex of the polypeptide according to the invention attached to the membrane and of the human serum antibody specifically recognizing it, which results in a localized coloration. at the level of said complex.
• Example 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 ⁇ g 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 ⁇ l per well).
• PBS polyoxyethylene sorbitan
• TMB tetrabenzimidine substrate
• 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.
• 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 children with pneumonitis, diagnosed as negative for 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 or witnesses.
• SEQ ID NO: 20 1 (5.0%) 20 20 (100.0%)
• 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.
• the 2A9, 6F2 and 10E2 proteins have poor performance (specificity and / or sensitivity) and can not be used in the serological diagnosis of M. pneumoniae infection in children or adults.
• the panel of samples tested consisted of two or three sera taken at different intervals from adult and pediatric patients whose M. pneumoniae infection was diagnosed by the demonstration of a positive IgM response by ELISA, and / or or the demonstration of a positive titre after a complement fixation test (RFC test) and / or by the detection of the microorganism by culture of samples and / or amplification by PCR.
• This panel is called the "Serum Monitoring Panel”.
• the panel of control individuals consists of sera of children and adults with pneumonitis, diagnosed negative for a M. pneumoniae infection according to the prior art.
• Tables 5 and 6 respectively show the results obtained according to the invention for the polypeptide 10B2 (SEQ ID NO: 2) with the monitoring panel described above and compare them with the results obtained with usual tests described in the prior art.
• Table 5 Numerical values and analysis of the ELISA test using the 10B2 polypeptide according to the invention and anti-IgM as secondary antibodies in the "Monitoring sera panel".
• the results show that the sensitivity of the IgM test using the 10B2 polypeptide according to the invention reaches 85.7% (18 sera of 21 patients detected) for a specificity of 94. , 8% (values obtained with the sera of the panel of control individuals not shown).
• the polypeptide allows the monitoring the serological status of the patient by observing the seroconversions (appearance of specific antibodies in response to the 10B2 antigen between two samples) as for example in patients 2, 5, and 6.
• Table 6 Comparison of the diagnostic performance (detection sensitivity) obtained with the IgM ELISA test using the 10B2 polypeptide according to the invention with those obtained with current diagnostic techniques described in the prior art as a commercial IgM ELISA test, the RFC test, culture or amplifications by PCR from samples.
• results show that the sensitivity of the ELISA test using the 10B2 polypeptide (SEQ ID No. 2) according to the invention is better than that of other tests described in the prior art (commercial ELISA test, RFC test, culture, PCR ) to diagnose M. pneumoniae infection in the panel of sera from patients studied. It is thus demonstrated, on the one hand, the existence, in humans, of a significant antibody response (the probability associated with a test of ⁇ 2 is less than 0.05) vis-à-vis the identified polypeptides according to the invention during M. pneumoniae infections and, on the other hand, that the proteins 10B2, 10A2, 10A1, 14G5, 15C6, 15E6, 14E5, 15D6 and 7D4 are relevant for the serological diagnosis of this type of infection. infection, through pathologies such as pneumonia.

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