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  Polypeptide nucleic sequences exported from mycobacteria, vectors comprising same and uses for diagnosing and preventing tuberculosisUSPTO Application #: 20070015173Title: Polypeptide nucleic sequences exported from mycobacteria, vectors comprising same and uses for diagnosing and preventing tuberculosis Abstract: Purified polynucleotides and polypeptides, and cells of M. smegmatis, M. bovis, M. bovis BCG, or M. africanum are provided. (end of abstract) Agent: Finnegan, Henderson, Farabow, Garrett & Dunner LLP - Washington, DC, US Inventors: Brigitte Gicquel, Denis Portnoi, Eng-Mong Lim, Vladimir Pelicic, Agnes Guigueno, Yves Goguet De La Salmoniere USPTO Applicaton #: 20070015173 - Class: 435006000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic Acid The Patent Description & Claims data below is from USPTO Patent Application 20070015173. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] The subject of the invention is novel recombinant screening, cloning and/or expression vectors which replicate in mycobacteria. Its subject is also a set of sequences encoding exported polypeptides which are detected by fusions with alkaline phosphatase and whose expression is regulated (induced or repressed) or constitutive during the ingestion of mycobacteria by macrophages. The invention also relates to a polypeptide, called DP428, of about 12 kD which corresponds to an exported protein found in mycobacteria belonging to the Mycobacterium tuberculosis complex. The invention also relates to a polynucleotide comprising a sequence encoding this polypeptide. It also relates to the use of the polypeptide or of fragments thereof and of the polynucleotides encoding the latter (or alternatively the polynucleotides complementary to the latter) for the production of means for detecting in vitro or in vivo the presence of a mycobacterium belonging to the Mycobacterium tuberculosis complex in a biological sample or for the detection of reactions of the host infected with these bacterial species. The invention finally relates to the use of the polypeptide or of fragments thereof as well as of the polynucleotides encoding the latter as means intended for the preparation of an immunogenic composition which is capable of inducing an immune response directed against the mycobacteria belonging to the Mycobacterium tuberculosis complex, or of a vaccine composition for the prevention and/or treatment of infections caused by mycobacteria belonging to said complex, in particular tuberculosis. [0002] The aim of the present invention is also to use these sequences (polypeptide and polynucleotide sequences) as target for the search for novel inhibitors of the growth and multiplication of mycobacteria and of their maintenance in the host, it being possible for these inhibitors to serve as antibiotics. [0003] The genus Mycobacterium, which comprises at least 56 different species, includes major human pathogens such as M. leprae and M. tuberculosis, the agents responsible for leprosy and tuberculosis, which remain serious public health problems worldwide. [0004] Tuberculosis continues to be a public health problem in the world. At present, this disease is the cause of 2 to 3 million deaths in the world and about 8 million new cases are observed each year (Bouvet, 1994). In developed countries, M. tuberculosis is the most common cause of mycobacteria infections. In France, about 10,000 new cases appear per year and, among the notifiable diseases, it is tuberculosis which comprises the highest number of cases. Vaccination with BCG (Bacille Calmette-Guerin), an avirulent strain which is derived from M. bovis and which is widely used as a vaccine against tuberculosis, is far from being effective in all populations. This efficacy varies from about 80% in western countries such as England, to 0% in India (results of the last vaccination trial in Chingleput., published in 1972 in Indian J. Med. Res.). Furthermore, the appearance of M. tuberculosis strains which are resistant to antituberculars and the increased risk in immunosuppressed patients, patients suffering from AIDS, of developing tuberculosis, make the development of rapid, specific and reliable methods for the diagnosis of tuberculosis and the development of novel vaccines necessary. For example, an epidemiological study carried out in Florida, and of which the results were published in 1993 in AIDS therapies, showed that 10% of the AIDS patients are affected by tuberculosis at the time of the AIDS diagnosis or 18 months before it. In these patients, tuberculosis appears in 60% of cases in a form which is disseminated and therefore nondetectable by conventional diagnostic criteria such as pulmonary radiography or the analysis of sputum. [0005] Currently, a certainty on the diagnosis provided by the detection of bacilli which can be cultured in a sample obtained from a patient is obtained in only less than half of the tuberculosis cases, even in the case of pulmonary tuberculosis. The diagnosis of tuberculosis and of the other related mycobacteria is therefore difficult to carry out for various reasons: mycobacteria are often present in a small quantity, their generation time is very long (24 h for M. tuberculosis) and they are difficult to culture (Bates et al., 1986). [0006] Other techniques can be used in clinical medicine to identify a mycobacterial infection: [0007] a) The direct identification of microorganisms under a microscope; this technique is rapid, but does not allow the identification of the mycobacterial species observed and lacks sensitivity (Bates, 1979). [0008] Cultures, when they are positive, have a specificity approaching 100% and allow the identification of the mycobacterial species isolated; however, as specified above, the growth of mycobacteria in vitro is long (can only be carried out in 3 to 6 weeks of repeated cultures (Bates, 1979; Bates et al., 1986)) and expensive. [0009] b) Serological techniques are found to be useful under certain conditions, but their use is sometimes limited by their low sensitivity and/or specificity (Daniel et al., 1987). [0010] c) The presence of mycobacteria in a biological sample can also be determined by molecular hybridization with DNA or RNA using oligonucleotide probes which are specific for the sequences tested for (Kiehn et al., 1987; Roberts et al., 1987; Drake et al., 1987). Several studies have shown the advantage of this technique for the diagnosis of mycobacterial infections. The probes used consist of DNA, ribosomal RNA or DNA fragments from mycobacteria which are obtained from gene banks. The principle of these techniques is based on the polymorphism of the nucleotide sequences of the fragments used or on the polymorphism of the adjacent regions. In all cases, they require the use of cultures and are not directly applicable to biological samples. [0011] The low quantity of mycobacteria present in a biological sample and consequently the low quantity of target DNA to be detected in this sample can require the use of a specific amplification in vitro of the target DNA before its detection with the aid of the nucleotide probe and using in vitro amplification techniques such as PCR (polymerase chain reaction). The specific amplification of the DNA by the PCR technique can constitute the first stage of a method for detecting the presence of a mycobacterial DNA in a biological sample, the actual detection of the amplified DNA being carried out in a second stage with the aid of an oligonucleotide probe capable of specifically hybridizing with the amplified DNA. [0012] A test for the detection of mycobacteria belonging to the Mycobacterium tuberculosis complex, by sandwich hybridization (test using a capture probe and a detection probe) was described by Chevrier et al. in 1993. The Mycobacterium tuberculosis complex is a group of mycobacteria which comprises M. bovis-BCG, M. bovis, M. tuberculosis, M. africanum and M. microti. [0013] A method for the detection of low quantities of mycobacteria, belonging to the tuberculosis complex, by gene amplification and direct hybridization on biological samples has been developed. Said method uses the insertion sequence IS6110 (European Patent EP 0,490,951 B1). Thierry et al. described in 1990 a sequence which is specific to the Mycobacterium tuberculosis complex and which is called IS6110. Some authors have proposed specifically amplifying the DNA obtained from Mycobacterium using nucleic primers in an amplification method, such as the polymerase chain reaction (PCR). Patel et al. described in 1990 the use of several nucleic primers chosen from a sequence known as a probe in the identification of M. tuberculosis. However, the length of the fragments obtained using these primers was different from the expected theoretical length and several fragments of variable size were obtained. Furthermore, the authors observed the absence of hybridization of the amplified products with the plasmid which served to determine the primers. These results indicate that these primers might not be appropriate in the detection of the presence of M. tuberculosis in a biological sample and confirm the critical nature of the choice of the primers. The same year, J. L. Guesdon and D. Thierry described a method for the detection of M. tuberculosis, having a high sensitivity, by amplification of an M. tuberculosis DNA fragment located within the IS6110 sequence (European Patent EP 461,045) with the aid of primers generating amplified DNA fragments of constant length, even when the choice of the primers led to the amplification of long fragments (of the order of 1000 to 1500 bases) where the risk of interruption of the polymerization is high because of the effects of the secondary structure of the sequence. Other primers specific for the IS6110 sequence are described in European Patent No. EP-0,490,951. [0014] The inventors have shown (unpublished results) that some clinical isolates of Mycobacterium tuberculosis lacked the insertion sequence IS6110 and could therefore not be detected with the aid of oligonucleotides specific for this sequence which could thus lead to false-negative diagnostic results. These results confirm a similar observation made by Yuen et al. in 1993. The impossibility of detecting these pathogenic strains which are potentially present in a biological sample collected from a patient is thus likely to lead to diagnostic difficulties or even to diagnostic errors. The availability of several sequences specific for the tubercule bacillus, within which primers appropriate for amplification will be chosen, is important. The DP428 sequence described here may be used. [0015] M. bovis and M. tuberculosis, the causative agents of tuberculosis, are facultative intracellular bacteria. [0016] These agents have developed mechanisms to ensure their survival and their replication inside macrophage, one of the cell types which is supposed to eradicate invasion by microorganisms. These agents are capable of modulating the normal development of their phagosome and of preventing them from becoming differentiated into an acidic compartment rich in hydrolase (Clemens, 1979; Clemens et al., 1996; Sturgill-Koszycki et al., 1994 and Xu et al., 1994). However, this modulation is only possible if the bacterium is alive inside the phagosome, suggesting that compounds which are actively synthesized and/or secreted inside the cell are part of this mechanism. Exported proteins are probably involved in this mechanism. Despite major health problems linked to these pathogenic organisms, little is known on their exported and/or secreted proteins. SDS-PAGE analyses of M. tuberculosis culture filtrate show at least 30 secreted proteins (Altschul et al., 1990; Nagal et al., 1991 and Young et al., 1992). Some of them have been characterized, their genes cloned and sequenced (Borremans et al., 1989; Wiker et al., 1992 and Yamaguchi et al., 1989). Others, although being immunodominant antigens of major importance for inducing a protective immunity (Anderson et al., 1991 and Orme et al., 1993), have not been completely identified. In addition, it is probable that many exported proteins remain attached to the cell membrane and are consequently not present in the culture supernatants. It has been shown that the proteins located at the outer surface of various pathogenic bacteria, such as the 103 kDa invasin of Yersina Pseudotuberculosis (Isberg et al., 1987) or the 80 kDa internalin of Listeria monocytogenes (Gaillard et al., 1991 and Dramsi et al., 1997) play an important role in the interactions with the host cells and, consequently, in the pathogenicity as well as in the induction of protective responses. Thus, a protein which is bound to the membrane would be important for the M. tuberculosis infection as well as for the induction of a protective response against this infection. These proteins could certainly be of interest for the preparation of vaccines. [0017] Recently, the adaptation, to mycobacteria, of a genetic methodology for the identification and the phenotypic selection of export proteins has been described (Lim et al., 1995). This method uses E. coli periplasmic alkaline phosphatase (PhoA). A plasmid vector was constructed which allows the fusion of genes between a truncated PhoA gene and genes encoding exported proteins (Manoil et al., 1990). [0018] Using this method, it has been possible to identify an M. tuberculosis gene (erp (Berthet et al., 1995)) exhibiting homologies with a 28 kDa exported protein of M. leprae, which is a frequent target of humoral responses of the lepromatous form of leprosy. A protein having amino acid motifs which are characteristic of plant desaturase (des) has also been characterized by the technique of fusion with PhoA. [0019] However, this genetic method for identifying exported proteins does not make it possible to easily evaluate the intracellular expression of the corresponding genes. Such an evaluation is of crucial importance both for selecting good candidate vaccines and for understanding the interactions between bacteria and their host cells. The induction of the expression of virulence factor through pathogenic target cell contact has been described. It is the case, for example, for the Yersinia pseudotuberculosis Yops virulence factors (Petersson et al., 1996). Shigella, upon contact with the target cells, releases the Ipa proteins into the culture medium, and Salmonella synthesizes novel surface structures. [0020] Taking into account the preceding text, a great need currently exists for developing novel vaccines against pathogenic microbacteria as well as novel specific, reliable and rapid diagnostic tests. These developments require the designing of even more efficient specific tools which make it possible, on the one hand, to isolate or to obtain sequences of novel specific, in particular immunogenic, polypeptides, and, on the other hand, to better understand the mechanism of the interactions between bacteria and their host cells such as in particular the induction of the expression of virulence factor. This is precisely the object of the present invention. [0021] The inventors have defined and produced, for this purpose, novel vectors allowing the screening, cloning and/or expression of mycobacterial DNA sequences so as to identify, among these sequences, nucleic acids encoding proteins of interest, preferably exported proteins, which may be located on the bacterial membrane, and/or secreted proteins, and to identify among these sequences those which are induced or repressed during infection (intracellular growth). DESCRIPTION [0022] The present invention describes the use of the reporter gene phoA in mycobacteria. It makes it possible to identify systems for expression and export in a mycobacterial context. Many genes are only expressed in such a context, which shows the advantage of the present invention. During the cloning of DNA segments of strains of the M. tuberculosis complex fused with phoA into another mycobacterium such as M. smegmatis, the beginning of the gene, its regulatory regions and its regulator will be cloned, which will make it possible to observe a regulation. If this regulation is positive, the cloning of the regulator will constitute an advantage for observing the expression and the export. [0023] In the context of the invention, mycobacterium is understood to mean all the mycobacteria belonging to the various species listed by Wayne L. G. and Kubica G. P. (1980). Family Mycobacteriaceae in Bergey's manual of systematic bacteriology, J. P. Butler Ed. 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