| PATENT ASSIGNEE'S COUNTRY | FRance |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 09.05.2000 |
| PATENT TITLE |
Mycobacterium proteins and applications |
| PATENT ABSTRACT |
Mycobacterium proteins, in particular those of M. bovis, having molecular weights between approximately 44.5 and 47.5 kD. These proteins can have molecular weights of approximately 45 kD or 47 kD and isoelectric pH of approximately 3.7 (45 and 47 kD proteins) and 3.9 (47 kD proteins). These proteins or hybrid proteins containing a part of their sequences can be used as vaccines or as drugs, or for the detection and monitoring of tuberculosis in particular in man and in cattle. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | November 30, 1994 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED |
Fifis et al. Infect. & Immun. Mar. 1991. 59(3):800-807. DeBruyn et al. Infect. & Immun. 1987.55(1):242-252. Romain et al. Feb. 1993. Infection & Immunity 61(2):742-750. Carlin et al. Aug. 1992. Infection & Immunity 60(8): 3136-3142. International Preliminary Examination Report for PCT/FR 92/00508 Dated Aug. 27, 1993 with English Translation. International Preliminary Examination Report for PCT/FR 92/00508 Dated Apr. 7, 1993. International Search for PCT/FR 92/00508 Dated Sep. 11, 1992. Search Report for FR 9106970 dated Jan. 23, 1992. Miura et al, "Comparative Studies with Various Substrains of Mycobacterium Bovis BCG on the Production of an Antigenic Protein, MPB70", Infection and Immunity, vol. 39, (1983), pp. 540-545. Abou-Zeid et al, "Characterization of the Secreted Antigens of Mycobacterium Bovis BCG: Comparison of the 46-Kilodalton Dimeric Protein with Proteins MPB64 and MPB70", Infection and Immunity, vol. 55, (1987), pp 3213-324. |
| PATENT CLAIMS |
We claim: 1. A protein isolated from Mycobacterium and having a molecular weight in the presence of SDS of between about 44.5 and 47.5 kD, wherein said protein is recognized by antibodies obtained by immunization with live Mycobacterium bovis bacilli, by the antibodies from tubercular patients and by an antibody secreted by a hybridoma deposited under No. I-1081 at the CNCM or a hybridoma line deposited under No. I-1483 at the CNCM, and is not recognized by the antibodies obtained by immunization with heat-killed Mycobacterium bovis bacilli or by antibodies from healthy patients or patients affected by a disease other than tuberculosis. 2. The protein of claim 1, wherein said protein is isolated from Mycobacterium bovis. 3. The protein of claim 2, wherein said protein has a molecular weight of approximately 45 kD. 4. The protein of claim 2, wherein said protein has a molecular weight of approximately 47 kD. 5. The protein of claim 2, having a pHi of approximately 3.7. 6. The protein of claim 2, having a pHi of approximately 3.9. 7. The protein of claim 4, wherein said protein has an NH.sub.2 terminal with the following sequence (SEQ ID NO:1): ALA-PRO-GLU-PRO-ALA-PRO-PRO-VAL-PRO-PRO-ALA-ALA- 1 2 3 4 5 6 7 8 9 10 11 12 - ALA-ALA-PRO PRO ALA 13 14 15 16 17. 8. The protein of claim 2, wherein said protein has an amino-acid composition expressed by frequency for PRO of approximately 21.9%, for ASN/ASP approximately 10.6%, for THR approximately 5.4%, for SER approximately 5%, for GLN/GLU approximately 6%, for GLY approximately 7.4%, for ALA approximately 19.2%, for VAL approximately 5.8%, for ILE approximately 2.3%, for LEU approximately 4.7%, for TYR approximately 2.2%, for PHE approximately 2.2%, for LYS approximately 2.9% and/or for ARG approximately 2.5%. 9. The protein of claim 2, wherein said protein is present in a culture medium of Mycobacterium bacilli. 10. A method for detecting or monitoring the development of tuberculosis in man or cattle, comprising assaying a biological sample from said man or cattle for antibodies which bind specifically to a protein isolated from Mycobacterium and having a molecular weight in the presence of SDS between about 44.5 and 47.5 kD, wherein said protein is recognized by antibodies obtained by immunization with live Mycobacterium bovis bacilli, by the antibodies from tubercular patients and by an antibody secreted by a hybridoma deposited under No. I-1081 at the CNCM or a hybridoma line deposited under No. I-1483 at the CNCM, and is not recognized by the antibodies obtained by immunization with heat-killed Mycobacterium bovis bacilli or by antibodies from healthy patients or patients affected by a disease other than tuberculosis; detecting the presence of said antibodies in the biological sample; and correlating the presence of said antibodies in the biological sample with the development of tuberculosis. 11. A process for the detection and monitoring of the development of tuberculosis in man and in cattle comprising the steps of: a) bringing a protein according to claim 1 into contact with a serum sample of a patient under conditions sufficient to obtain a complex between said protein and any antibody present is said serum sample; and b) detecting said complex. |
| PATENT DESCRIPTION |
The present invention relates to Mycobacterium proteins, in particular those of M. bovis, having molecular weights between approximately 44.5 and 47.5 kD and the nucleotide sequences coding for these proteins. The present invention also relates to those protein fractions obtained from cultures of Mycobacterium bovis, showing a specific immunological reactivity towards anti-tuberculosis antibodies. It also relates to the use of these proteins and fractions for the detection and monitoring of tuberculosis and as vaccines. Tuberculosis continues to be a public health problem throughout the world. The annual number of deaths directly related to tuberculosis is around 3 million and the number of new cases of tuberculosis is around 15 million. This number of deaths due to tuberculosis is high even for the developed countries; for example in France it is of the order of 1500 per year, a figure which is certainly underestimated by a factor of 2 or 3 if Roujeau's assessments of the differences between official figures and the results of systematic autopsies are taken into account. The recent increase in tuberculosis cases, or at least the leveling-off of the decrease in the frequency of this disease, must be considered in correlation with the development of the HIV/AIDS epidemic. In total tuberculosis remains the leading infectious disease in terms of frequency in France and the developed countries, but above all in the developing countries for which it constitutes the principal source of human loss related to a single disease. At present, a definite diagnosis made by the demonstration of cultivable bacilli in a sample taken from the patient is only obtained in less than half the cases of tuberculosis. Even for pulmonary tuberculosis, which represents 80 to 90% of the tuberculosis cases, and which is the form of the disease for which the detection of the bacilli is the easiest, the examination of sputum is only positive for less than half the cases. The development of the most sensitive techniques such as PCR (Polymerized Chain Reaction), always comes up against the necessity for obtaining a sample. Since women and children do not habitually spit, sampling for infants frequently requires specialized medical intervention (for example ganglionic biopsy or sampling by lumbar puncture of the cephalo-rachidian fluid). In other respects, inhibitions of the PCR reaction itself exist, of a type such that a sample can be unusable by this technique because of the impossibility of controlling their origins. Finally, the conventional bacteriological diagnosis, microscopic examination and culture, because of its sensitivity limits (at the best of the order of 10.sup.4 to 10.sup.5 bacilli in the sample) requires that there has already been a relatively substantial development of bacilli and thus of the disease. The detection of specific antibodies directed against Mycobacrerium tuberculosis should thus be of assistance in the diagnosis of the common forms of the disease for which the detection of the bacilli themselves is difficult or impossible. Successive generations of research workers have attempted to perfect a serological diagnosis technique for tuberculosis. From Ardoing (C.r. hebd. Seanc. Acad. Sci. Paris; 1898: 126: 1398-1401) to Middlebrook and Dubos (J. Exp. Med. 1948, 88: 521-528), the preparations used for this diagnosis have been very little or not purified, the effort being directed above all towards an increase in sensitivity and not specificity. Recently again techniques tending to increase only the sensitivity have been suggested using a technique of either the ELISA or RIA type. The more recent work of Daniel and Janicki (Microbiol. Rev. 1978, 42; 84-113) or Wiker et al. (Scand. J. Immunol. 1988, 27: 223-239) has shown the complexity of the mycobacterial antigens. Following this work, attempts have been made to define the principal antigens able to be used for diagnostic purposes (Chan et al., Am. Rev. Respir. Dis. 1990, 142: 385-390). A serological test has as a result been commercially available (ANDA) for 3 or 4 years. It uses an extremely complex antigen, poorly characterized biochemically, the A60 of Cocito and Vanlinden (Clin. Exp. Immunol. 1986, 66: 262-272). The specificity and sensitivity of this test are poor. It is not of great help in diagnosis and has been highly criticized if not rejected by numerous biologists who have used it widely. Well characterized antigens, corresponding to quantitatively abundant proteins, have also been tested for their specificity and sensitivity for serological diagnosis. The .alpha. antigen of approximately 35 kD was first purified in 1965; it represents 25 to 35% of the proteins present in the culture medium of numerous strains of M. tuberculosis, M. kansasii or BCG (Fukui et al. Biken J. 1965, 8: 189-199). An antigen of 64 kD has also been purified, representing more than 50% of the culture medium proteins, when these are grown in the absence of zinc (De Bruyn et al. J. Gen. Microbiol. 1981, 124: 353-357). A second molecule has also been purified, representing 25 to 30% of the proteins in the same media. This molecule of 32 kD is very similar to the a antigen already described, but is nevertheless different (De Bruyn et al. Microbiol. Pathogenesis 1987, 2: 351-366). Similarly, a molecule of 16 kD has been purified, and its presence has been observed in the culture medium of certain BCG (NCG Tokyo) and M. bovis strains and its absence or low level for other strains (BCG Copenhague, Pasteur) (Harboe and Nagai, Am. Rev. Respir. Dis. 1984, 139: 444-452). The selection criteria for the proteins used by these authors have thus been in the first place biochemical criteria. The proteins have only been subsequently tested for their capacity to detect infection by tuberculosis. Representative results of this approach thus show that the antibody response directed against the 64 kD thermal shock protein of mycobacteria is high in 80% of patients affected by tuberculosis, but also in 30% of infants affected by whooping cough (Thole et al. Infect. Immun. 1987, 55: 1466-1475). The use of molecular biological techniques for gene cloning where the product is recognized by a polyclonal antibody of restricted specificity or a monoclonal antibody, has led to more or less the same results. In this case, the antibodies which have been used as probes, have been prepared against the antigens present in bacterial extracts or on killed bacteria. These extracts or bacteria having been as a general rule injected into Balb/C mice, the repertory of responses obtained is limited to the response of the mouse of this single cloning line, submitted moreover to extremely similar experimental protocols. Having thus been recognized by certain monoclonal antibodies, such as the 65 kD or the 32 kD, the molecules have already been purified or are in the course of purification by a classical biochemical approach. New molecules such as the 70 kD or 19 kD have also been detected by this technique. But when used for detection purposes, these molecules do not allow unambiguous distinction between patients affected by tuberculosis and normal subjects or those with other infectious diseases. In recent work the purification of mycobacterial antigens has been attempted using a mixture of serums from tuberculosis patients to make an immuno-adsorbent allowing partial purification of the principal antigens recognized by the patients (Thongkrajai et al. J. Med. Microbiol. 1989, 30: 101-104). The techniques reported in the prior art are thus mostly based on the preliminary isolation of proteins through their biochemical properties. It is not until after this isolation that the authors have tested the capacity of these proteins to detect those individuals affected by tuberculosis. In the work leading up to the present invention, another method has been chosen to select the antigens representative of tuberculosis infection. According to the invention, the work has been directed towards the unambiguous selection of the antigens representative of tubercular infection by the use of serums originating from patients affected by tuberculosis or from guinea pigs immunized with live bacilli. This method, which is distinguished from those experiments described in the prior art, has allowed the isolation of antigens representative of tuberculosis, permitting the unambiguous detection of patients affected by this disease. The present invention thus relates to the proteins of Mycobacterium and in particular of M. bovis having a molecular weight of between approximately 44.5 and 47.5 kD. These proteins can have molecular weights of approximately 45 kD or approximately 47 kD, within limits of error of .+-.10%, and isoelectric pH (pHi) of approximately 3.7 (proteins 45 and 45 kD) and 3.9 (proteins of 47 kD), with pHi limits of error of .+-.0.2. The 10% error in the molecular weight determination is in particular due to variations in results according to the determination kit used (LMW Electrophoresis Calibration Kit, Ref. 17-0446-01, Pharmacia). These proteins can also possess an amino-acid composition expressed by frequency for PRO of approximately 21.9%, for ASN/ASP approximately 10.6%, for THR approximately 5.4%, for SER approximately 5%, for GLN/GLU approximately 6%, for GLY approximately 7.4%, for ALA approximately 19.2%, for VAL approximately 5.8%, for ILE approximately 2.3%, for LEU approximately 4.7%, for TYR approximately 2.2%, for PHE approximately 2.2%, for LYS approximately 2.9%, and/or for ARG approximately 2.5%. The 47 kD protein species can have an NH.sub.2 terminal with the following sequence (SEQ ID N.sup.o 1): ALA-PRO-GLU-PRO-ALA-PRO-PRO-VAL-PRO-PRO-ALA-ALA- 1 2 3 4 5 6 7 8 9 10 11 12 - ALA-ALA-PRO-PRO-ALA 13 14 15 16 17 The present invention also relates to a hybridoma line deposited on the Apr. 12, 1991 under the N.sup.o I-1081 as part of the Collection Nationale de Culture des Microorganismes (CNCM) of the Institut Pasteur, and to a hybridoma line deposited on Oct. 12, 1994 under the N.sup.o I-1483 as a part of the CNCM, and the antibodies secreted by these lines. The proteins described above also have the property of being recognized by antibodies present in the serum of patients affected by tuberculosis or of animals able to be affected by tuberculosis, by certain antibodies obtained by immunization of guinea pigs with live M. bovis bacilli, or by an antibody secreted by the aforementioned hydridoma lines N.sup.o I-1081 or I-1483 and of not being recognized by antibodies obtained by immunization of guinea pigs with M. bovis bacilli killed by heat treatment or by antibodies of healthy patients or those affected by a disease other than tuberculosis. These proteins are also characterized by the fact that they can be present in the culture medium. According to a particular use of the invention, an antigenic determinant (epitope) originating from a biological agent other than M. bovis can also be grafted onto one of the proteins defined above. Hybrid proteins are thus obtained of which the sequence includes the whole or part of the sequence of the proteins described above and a sequence corresponding to an antigenic determinant. This determinant can be of various types and can in particular be a fragment of a protein or glycoprotein antigen, in order to obtain immunogenic compositions able to induce the synthesis of antibodies directed against these multiple antigenic determinants. The use of bifunctional bridging agents such as glutaraldehyde or benzoquinone or N-bromosuccinimide, well known for their ability to interlink protein chains, or hydrazide allowing the linking of glycosyl residues with proteins, can be used for the formation of hybrid molecules. These hybrid molecules can be composed in part of a carrier molecule (45-47 kD complex), associated with one or several antigenic determinants or antigen fragments, for example diphtheria toxin or fragments thereof, tetanus toxin, the surface antigen of hepatitis B virus, poliomyelitis virus VP1 antigen. The synthesis processes for hybrid molecules encompass the methods used in genetic engineering to construct DNA hybrids coding for the protein or peptide sequences required. Such proteins can thus induce immunization against proteins or protein fragments corresponding to the antigenic determinants not present on the M. bovis proteins. The invention also relates to the oligonucleotides, RNA or DNA, coding for the proteins defined above. The present invention relates in addition to the protein fractions obtained from Mycobacterium cultures and in particular from M. bovis by a process including at least the following stages elimination of the bacteria from the culture medium by filtration, passage of the filtrate over a molecular sieve, and division of the eluate into fractions, and selection of the fractions by determination of their reactivity towards specific tuberculosis antibodies. The fractions obtained by filtration over a molecular sieve can also be subjected to ion exchange chromatography and optionally to reversed phase chromatography. The present invention also relates to the application of the proteins or the protein fractions or antibodies such as those defined above for the detection and monitoring of tuberculosis in particular in humans and bovines. Such detection can in particular be carried out by the Western Blot (immuno-imprint) method or an immunoenzymological method (ELISA) or by a radioimmunological (RIA) method, by use of a measurement pack or kit, containing these proteins as well as in particular the buffers allowing the immunological reaction to be carried out and in addition substances allowing this to be revealed. The present invention also relates to vaccines or drugs containing at least one protein, one protein fraction, or one antibody such as those defined above. Vaccines containing nongrafted proteins can be used to immunize individuals against tuberculosis. The proteins carrying an antigenic determinant originating from a biological agent other than M. bovis can be used in the framework of immunization against other diseases. As an indication, from 50 to 500 .mu.g of protein can be used for an individual dose, or from 10.sup.5 to 10.sup.6 recombinant bacteria/individual by intradermic methods. The present invention also relates to a pharmaceutical composition containing at least a pharmaceutically effective quantity of a protein, protein fraction or antibody, such as those defined above, in combination with pharmaceutically acceptable diluents or adjuvants. In another respect, the present invention relates to the use of proteins, protein fractions or antibodies, such as those already defined above, for the manufacture of a drug for the treatment or prevention of tuberculosis. The present invention is illustrated, without in any way being limited, by the following examples of implementation and with reference to the annexed drawings in which: FIG. 1 shows the optical density (OD) profile at 220 and at 280 nanometers of the molecular filtration (Si 300) of the M. bovis culture medium. FIG. 2 shows the optical density profile at 220 nanometers of the separation on ion exchange column (DEAE) of molecules originating from fraction 2 obtained during the above molecular filtration. FIG. 3 shows the optical density profile at 220 nanometers of reversed phase column chromatography of fraction 1 resulting from the ion exchange chromatography. FIGS. 4A and 4B are photographs of PVDF membranes in the presence respectively of a mixture of serums from guinea pigs immunized with dead (A) or live (B) bacilli. FIG. 4C is an electrophoresis gel after coloration by Coomassie blue of the starting material (0) and of fractions obtained on the molecular sieve (1 to 6). Identical gels were transferred onto a PVDF membrane and revealed by serums from guinea pigs immunized by dead (4A) or live (4B) bacilli. FIGS. 5A and 5B show PVDF membranes corresponding to a gel obtained by migration of fractions obtained on ion exchange columns (1 to 3) and of fraction 2 obtained by molecular sieve filtration, the said membrane being placed in the presence of antibodies of serums from guinea pigs immunized respectively with dead (A) or live (B) bacilli. FIG. 5C represents the original gel after the transfers of FIGS. 5A and 5B, colored by Coomassie blue. FIGS. 6A and 6B show the imprint of gels on the membranes corresponding to the migration of fraction 1 obtained on ion exchange column (0) and the fractions obtained by reversed phase chromatography (1 to 5), placed in the presence of antibodies from serums from guinea pigs immunized respectively with dead (6A) and live (6B) bacilli. It should be noted that in fraction 1 of this purification stage, there is a contamination by the starting material due to too high a loading of the reversed phase chromatography column. FIGS. 7A to 7L show the imprints of gels on membranes obtained through electrophoresis of starting material (0), fractions obtained by molecular sieve filtration (1 to 6) and placed in the presence of individual serums from patients affected by tuberculosis (7A to 7F) corresponding respectively to serum from patients (N.sup.o 77, N.sup.o 115, N.sup.o 117, N.sup.o 108, N.sup.o 104, N.sup.o 105) or by individual serums originating from patients suffering from Borrelia infection (7G to 7K) or yersiniosis (7L). FIG. 8 shows the imprint of a gel on a PVDF membrane corresponding to the electrophoresis of fraction 5 obtained by reversed phase chromatography. This membrane was cut into approximately 3 mm strips, and each strip was placed in the presence of an individual serum (diluted 1/20) from a patient affected by tuberculosis (bands 1 to 14 corresponding respectively to patient N.sup.o 77, 104, 105, 108, 115, 117, 124, 131, 134, 123, 3a, 2g, 2d, and 2a), or patients suffering from Borrelia infection (bands 15 to 19), leptospirosis (bands 20 to 22), yersiniosis (bands 23 and 24), or brucellosis (bands 25 to 27). The double "a" marks correspond to a common artefact in this technique. FIG. 9 is a photograph of a two-dimensional gel colored with silver nitrate for proteins of 45-47 kD (fraction 5). FIGS. 10, 11 and 12 represent some immunoblottings of crude antigens preparations of M.bovis AN5 (track 1), M. avium (track 2), M.bovis BCG (track 3), M. tuberculosis H37Rv (track 4), M.kansaii (track 5), M. microti (track 6), M. smegmatis (track 7), M.xenopi (track 8) respectively incubated with monoclonal antibodies of lines I-1483 (FIG. 10), C.sub.13 (FIG. 11) and A.sub.5 A.sub.3 (FIG. 12) |
| PATENT EXAMPLES | This data is not available for free |
| PATENT PHOTOCOPY | Available on request |
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