Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...
Reexamination Certificate
1998-07-16
2002-08-20
Swartz, Rodney P (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Bacterium or component thereof or substance produced by said...
C435S006120, C435S091100, C435S091200, C435S253100, C435S863000, C530S350000, C536S024320, C536S024330
Reexamination Certificate
active
06436409
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is directed to a polynucleotide comprising an open reading frame coding for a polypeptide from
Mycobacterium tuberculosis,
named LHP capable of inducing an immune response in a host. lhp is placed under the control of its own regulation signals which are functional in mycobacteria, especially in mycobacteria belonging to the
Mycobacterium tuberculosis
complex and also in fast growing mycobacteria such as
Mycobacterium smegmatis
and also in
E. coli.
The
Mycobacterium tuberculosis
complex has its usual meaning, i.e. the complex of mycobacteria causing tuberculosis which are
Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium microti
and the vaccine strain
M. bovis
BCG.
The invention is also directed to the polypeptide LHP encoded by lhp and most preferably to suitable antigenic portions of LHP as well as to oligomeric polypeptides containing more than one unit of LHP or an antigenic portion of LHP. The invention concerns also immunogenic and vaccine compositions containing a polypeptide or an oligomeric polypeptide such as defined above or live recombinant attenuated mycobacteria transformed with a polynucleotide according to the present invention. The invention also concerns antibodies directed specifically against such polypeptides that are useful as diagnostic reagents. In another embodiment, the present invention is directed to a polynucleotide carrying the natural regulation signals of lhp which is useful in order to express heterologous proteins in mycobacteria as well as functionally active regulatory polynucleotides derived from said regulatory region. Finally, the present invention is directed to oligonucleotides comprising at least 12 consecutive nucleotides which are useful as reagents for detecting the presence of
Mycobacterium tuberculosis
in a biological sample.
2. Related Prior Art
Mycobacterium tuberculosis
and
M. bovis
cause tuberculosis, a disease which currently kills three million people each year. The virulence of pathogenic mycobacteria is associated with their ability to parasitize and survive within phagocytic cells. Little is known about mechanisms governing gene expression during the intracellular growth stage. This issue is of prime importance as the intracellular stage of pathogenic mycobacteria can be viewed as an adaptative process, involving transcriptional regulatory mechanisms. Mycobacterial genes affecting intracellular growth and virulence are being actively sought (Collins, 1996; Collins, 1995, Quinn, 1996). Using subtractive genomic hybridization between virulent
M. bovis
and the attenuated vaccine strain
M. bovis
BCG, Maheiras et al. (Maheiras et al., 1996) identified three regions of difference (RD1 to RD3). RD1 was detected in all strains of
M. tuberculosis
and
M. bovis
tested but is absent in all BCG substrains, suggesting that it may be an important determinant of virulence.
The or flC gene, encoding the early secreted antigenic target 6 kDa (ESAT-6) lies within RD1. The ESAT-6 protein is a major T-cell antigen which has been purified from
M. tuberculosis
short-term culture filtrates (Harboe et al., 1996; Sorensen et al., 1995). Purified ESAT-6 stimulates the production of gamma interferon from mice memory immune T lymphocytes and may contribute to the development of antituberculous immunity (Andersen et al., 1995; and U.S. Patent Application filed on Jun. 5, 1995).
The Mycobacterium genus encompasses more than 70 recognized bacterial species including
M. tuberculosis
and
M. leprae
, the agents of tuberculosis and leprosy respectively. The development of effective prophylactic vaccine and specific diagnostic reagents is a priority to control the extension of mycobacterial infections. In that context, mycobacterial protein antigens are extensively screened upon their ability to induce B- and T-cell reactivity. Obtaining purified proteins from slow growing pathogenic mycobacteria is labor-intensive and requires important containment facilities. Alternatively, many immunological studies of mycobacterial antigens have been conducted with
E. coli
—expressed recombinant molecules. However, problems related to lipopolysaccharide (LPS) contamination are frequently encountered. Moreover, post-translational modifications such as proteolytic processing, intern removal, lipid acylation and glycosylation of proteins have been reported to occur in mycobacteria. Such modification cannot be mimicked in
E. coli
and may influence dramatically the stability, antigenicity and the immunogenicity of the peptide chain. Thus, it was recently postulated that site-specific mannosylation protects the
M. tuberculosis
19 kDa lipoprotein antigen against proteolysis (Hermann. et al., 1996). Accordingly, there is a great need in the art of suitable protein expression systems allowing the preparation of mycobacterial immunogenic polypeptides that are useful for diagnostic and vaccine purposes.
SUMMARY OF THE INVENTION
Now, the inventors have discovered a polynucleotide carrying the regulatory expression signals of the ESAT-6 protein as well as an open reading frame coding for a new antigenic protein from
Mycobacterium tuberculosis
that they have named LHP.
The LHP polypeptide of the invention shares a great similarity with a
Mycobacterium tuberculosis
peptide described in the PCT Application No. WO 97/09429 or in the PCT Application No. WO 97/09428 (Corixa Corporation) a partial sequence of which is disclosed in those patent applications.
The present inventors have characterized the portions of the polynucleotide according to the invention that are functional in mycobacteria in order to allow the expression of LHP, as well as the expression of an heterologous polypeptide that is placed under the control of said regulatory region contained in the polynucleotide according to the present invention.
More specifically, the inventors have located the transcription initiation sites of the lhp/or flC operon using
M. tuberculosis
RNA and have precisely mapped the portions of the regulatory region of the lhp/or flC operon that are functional in bacteria in general, being functionally active in
E. coli
as well as in mycobacteria. Further, the inventors have mapped the portions of the polynucleotide according to the present invention that are functionally active in slow growing mycobacteria, such as bacteria belonging to the
Mycobacterium tuberculosis
complex, and in fast-growing mycobacteria, such as
M. smegmatis.
Further, the present inventors have used the functionally active portions of the regulatory region of the lhp/or flC operon for expressing a polypeptide heterologous with respect to said regulatory region.
In a specific embodiment, the present inventors have constructed a mycobacterial expression vector allowing production of recombinant proteins tagged by a stretch of six histidine. Such vector enables production of virtually any polypeptide in a mycobacterial context and allows easy purification of native proteins by immobilized metal affinity chromatography. Additionally, the availability of monoclonal antibody directed against the (His)6 polypeptide facilitates the detection of proteins for which no specific immune reagent are available. This system is very useful for biochemical and immunological characterization of mycobacterial proteins.
Accordingly, given its high level and constitutive expression of the regulatory polynucleotide according to the present invention in mycobacteria, said promoter is used to construct a novel mycobacterial expression/purification system.
This vector, designated pIPX30, allows versatile gene fusions to produce histidine-tagged proteins in mycobacteria. Additionally, the high affinity of polyhistidine for immobilized metal ions enables one-step chromatographic isolation of native, histidine-tagged polypeptides. As a validation of the system, the inventors have performed the expression of recombinant DES(Histidine)
6
M. tuberculosis
protein antigen and its immunodetection fr
Andersen Peter
Berthet Francois-Xavier
Gicquel Brigitte
Rasmussen Peter Birk
Institut Pasteur
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Swartz Rodney P
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