DNA molecule fragments encoding for cellular uptake of mycobacterium tuberculosis and uses thereof

Abstract

The present invention relates to a DNA molecule conferring on Mycobacterium tuberculosis an ability to enter mammalian cells and to survive within macrophages. Peptides, proteins, or polypeptides (e.g. the Mycobacterium cell entry protein or Mcep) encoded by this gene fragment are useful in vaccines to prevent infection by Mycobacterium tuberculosis, while the antibodies raised against these peptides, proteins, or polypeptides can be employed in passively immunizing those already infected by the organism. These proteins, peptides, polypeptides, and antibodies may be utilized in diagnostic assays to detect Mycobacterium tuberculosis in tissue or bodily fluids. The peptides, proteins, or polypeptides of the present invention can be associated with various other therapeutic materials, for administration to mammals, particularly humans, to achieve uptake of those materials by such cells. Synthetically constructed peptides based on the disclosed amino acid sequences exhibit the same mammalian cell uptake activity observed with Mcep.

What is claimed:  
     
         1 . An isolated DNA molecule conferring an ability to enter mammalian cells, wherein said DNA molecule is a fragment of the DNA molecule in  Mycobacterium tuberculosis  which confers on that organism an ability to enter mammalian cells.  
     
     
         2 . An isolated DNA molecule according to    claim 1   , wherein said DNA molecule encodes the amino acid sequence corresponding to SEQ. ID. No. 8 or SEQ. ID. No. 10.  
     
     
         3 . An isolated DNA molecule according to    claim 2   , wherein said DNA molecule corresponds to the nucleotide sequence corresponding to SEQ. ID. No. 7 or SEQ. ID. No. 9.  
     
     
         4 . An isolated DNA molecule according to    claim 1   , wherein said DNA molecule encodes a variant of the amino acid sequence corresponding to SEQ. ID. No. 8 or SEQ. ID. No. 10 with the variant being unchanged at its amino acid positions corresponding to the following amino acids in SEQ. ID. No. 4: positions 27 and 28, position 32, or position 38.  
     
     
         5 . An isolated peptide encoded by a DNA molecule according to    claim 1   .  
     
     
         6 . An isolated peptide according to    claim 5   , wherein the protein or polypeptide has an amino acid sequence corresponding to SEQ. ID. No. 8 or SEQ. ID. No. 10.  
     
     
         7 . An isolated peptide according to    claim 5   , wherein said DNA molecule has a nucleotide sequence corresponding to SEQ. ID. No. 7 or SEQ. ID. No. 9.  
     
     
         8 . An isolated peptide according to    claim 5   , wherein the peptide is a variant of the amino acid corresponding to SEQ. ID. No. 8 or SEQ. ID. No. 10 with the variant being unchanged at its amino acid positions corresponding to the following amino acids in SEQ. ID. No. 4: positions 27 and 28, position 32, or position 38.  
     
     
         9 . An isolated peptide according to    claim 5   , wherein said peptide is recombinant.  
     
     
         10 . An isolated peptide according to    claim 5   , wherein said peptide is purified.  
     
     
         11 . A method of vaccinating mammals against infection by  Mycobacterium tuberculosis  comprising: 
 administering an effective amount of the isolated peptide according to    claim 5    to mammals.    
     
     
         12 . A method according    claim 11    wherein said administering is oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intranasal.  
     
     
         13 . A recombinant DNA expression system comprising an expression vector into which is inserted a heterologous DNA according to    claim 1   .  
     
     
         14 . A recombinant DNA expression system according    claim 13   , wherein said heterologous DNA corresponds to the nucleotide sequence corresponding to SEQ. ID. No. 7 or SEQ. ID. No. 9.  
     
     
         15 . A recombinant DNA expression system according to    claim 13   , wherein said heterologous DNA is inserted into said vector in proper orientation and correct reading frame.  
     
     
         16 . A host cell incorporating a heterologous DNA according to    claim 1   .  
     
     
         17 . A host cell according to    claim 16   , wherein said heterologous DNA corresponds to the nucleotide sequence corresponding to SEQ. ID. No. 7 or SEQ. ID. No. 9.  
     
     
         18 . A host cell according to    claim 16   , wherein said heterologous DNA is inserted in a recombinant DNA expression system comprising an expression vector.  
     
     
         19 . A vaccine for preventing infection and disease of mammals by  Mycobacterium tuberculosis  comprising: 
 an isolated peptide according to    claim 5   ; and    a pharmaceutically-acceptable carrier.    
     
     
         20 . A vaccine according to    claim 19   , wherein said peptide is purified.  
     
     
         21 . A method of vaccinating mammals against infection by  Mycobacterium tuberculosis  comprising: 
 administering an effective amount of the vaccine according to    claim 19    to mammals.    
     
     
         22 . A method according    claim 21   , wherein said administering is oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intranasal.  
     
     
         23 . An isolated antibody or binding portion thereof or a probe against a peptide according to    claim 5   .  
     
     
         24 . An isolated antibody or binding portion thereof or probe according to    claim 23   , wherein said antibody is monoclonal or polyclonal.  
     
     
         25 . An isolated antibody or binding portion thereof or probe according to    claim 23   , wherein said antibody is specific for an antigenic determinant of said peptide encoded by a gene fragment conferring on  Mycobacterium tuberculosis  an ability to enter mammalian cells.  
     
     
         26 . A method of passively immunizing mammals infected with  Mycobacterium tuberculosis  comprising: 
 administering an effective amount of said antibody or binding portion thereof or probe according to    claim 23    to mammals infected with  Mycobacterium tuberculosis.      
     
     
         27 . A method according to    claim 26   , wherein said administering is oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intranasal.  
     
     
         28 . A composition for passively immunizing mammals infected with  Mycobacterium tuberculosis  comprising: 
 an isolated antibody or binding portion thereof or probe according to    claim 23   ; and    a pharmaceutically-acceptable carrier.    
     
     
         29 . A composition according to    claim 28   , wherein said antibody is monoclonal or polyclonal.  
     
     
         30 . A composition according to    claim 28   , wherein said antibody is specific for an antigenic determinant of said peptide encoded by a gene fragment conferring on  Mycobacterium tuberculosis  an ability to enter mammalian cells.  
     
     
         31 . A method of passively immunizing mammals infected with  Mycobacterium tuberculosis  comprising: 
 administering an effective amount of said composition according to    claim 28    to mammals infected with  Mycobacterium tuberculosis.      
     
     
         32 . A method according to    claim 31   , wherein said administering is. oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intranasal.  
     
     
         33 . A method for detection of  Mycobacterium tuberculosis  in a sample of tissue or body fluids comprising: providing a peptide according to    claim 5    as an antigen; 
 contacting the sample with the antigen; and  
 detecting any reaction which indicates that  Mycobacterium tuberculosis  is present in the sample using an assay system.  
 
     
     
         34 . A method according to    claim 33   , wherein the assay system is selected from the group consisting of an enzyme-linked immunosorbent assay, a radioimmunoassay, a gel diffusion precipitin reaction assay, an immunodiffusion assay, an agglutination assay, a fluorescent immunoassay, a protein A immunoassay, and an immunoelectrophoresis assay.  
     
     
         35 . A method for detection of  Mycobacterium tuberculosis  in a sample of tissue or body fluids comprising: providing an antibody or binding portion thereof or probe according to    claim 23   ; 
 contacting the sample with the antibody or binding portion thereof or probe; and    detecting any reaction which indicates that  Mycobacterium tuberculosis  is present in the sample using an assay system.    
     
     
         36 . A method according to    claim 35   , wherein the assay system is selected from the group consisting of an enzyme-linked immunosorbent assay, a radioimmunoassay, a gel diffusion precipitin reaction assay, an immunodiffusion assay, an agglutination assay, a fluorescent immunoassay, a protein A immunoassay, and an immunoelectrophoresis assay.  
     
     
         37 . A method for detection of  Mycobacterium tuberculosis  in a sample of tissue or body fluids comprising: providing a nucleotide sequence of the DNA molecule according to    claim 1    as a probe in a nucleic acid hybridization assay; 
 contacting the sample with the probe; and  
 detecting any reaction which indicates that  Mycobacterium tuberculosis  is present in the sample.  
 
     
     
         38 . A method for detection of  Mycobacterium tuberculosis  in a sample of tissue or body fluids comprising: 
 providing a nucleotide sequence of the DNA molecule according to    claim 1    as a probe in a gene amplification detection procedure;    contacting the sample with the probe; and    detecting any reaction which indicates that  Mycobacterium tuberculosis  is present in the sample.    
     
     
         39 . A product for uptake of materials into mammalian cells comprising: 
 a material for uptake by mammalian cells; and    a peptide according to    claim 5   , wherein said peptide is associated with said material.    
     
     
         40 . A product according to    claim 39   , wherein said peptide has an amino acid sequence corresponding to SEQ. ID. No. 8.  
     
     
         41 . A product according to    claim 39   , wherein said peptide has an amino acid sequence corresponding to SEQ. ID. No. 10.  
     
     
         42 . A product according to    claim 39   , wherein the peptide is a variant of the amino acid corresponding to SEQ. ID. No. 8 or SEQ. ID. No. 10 with the variant being unchanged at its amino acid positions corresponding to the following amino acids in SEQ. ID. No. 4: positions 27 and 28, position 32, or position 38.  
     
     
         43 . A product according to    claim 39   , wherein said peptide is purified.  
     
     
         44 . A product according to    claim 39   , wherein said material is selected from the group consisting of antibiotics, DNA fragments, anti-neoplastic agents, and mixtures thereof.  
     
     
         45 . A cellular uptake process comprising: 
 directing a material into mammalian cells with a peptide according to    claim 5   .    
     
     
         46 . A process according to    claim 45   , wherein said peptide has an amino acid sequence corresponding to SEQ. ID. No. 8.  
     
     
         47 . A process according to    claim 45   , wherein said peptide has an amino acid sequence corresponding to SEQ. ID. No. 10.  
     
     
         48 . A process according to    claim 45   , wherein the peptide is a variant of the amino acid corresponding to SEQ. ID. No. 8 or SEQ. ID. No. 10 with the variant being unchanged at its amino acid positions corresponding to the following amino acids in SEQ. ID. No. 4: positions 27 and 28, position 32, or position 38.  
     
     
         49 . A process according to    claim 45   , wherein said peptide is purified.  
     
     
         50 . A process according to    claim 45   , wherein said material is selected from the group consisting of antibiotics, DNA fragments, anti-neoplastic agents, and mixtures thereof.  
     
     
         51 . A product for uptake of materials into mammalian cells comprising: 
 a material for uptake by mammalian cells; and    a peptide according to    claim 5   , wherein said peptide is associated with said material.    
     
     
         52 . A cellular uptake process comprising: 
 directing a material into mammalian cells with a peptide according to    claim 5   .