DNA vaccines encoding heat shock proteins

Abstract

The present invention is related to recombinant constructs encoding heat shock proteins or active fragments thereof that are effective in treating T cell mediated inflammatory autoimmune diseases by DNA vaccines. The treatment with the recombinant constructs of the present invention provides long-term expression of specific heat shock proteins for fragments thereof. In one embodiment, the present invention is related to a recombinant construct of a nucleic acid sequence encoding HSP60, HSP70 or HSP90. The present invention also is related to a recombinant construct of a nucleic acid sequence selected from amino acids 1-140 of HSP60, amino acids 130-260 of HSP60 and amino acids 31-50 of HSP60. Another aspect of the present invention is related to an active fragment of HSP60 corresponding to amino acids 31-50 of HSP60 effective in treating arthritis.

1 . A DNA vaccine for treating a T cell-mediated inflammatory autoimmune disease comprising a recombinant construct, the recombinant construct comprising a nucleic acid sequence encoding a mammalian heat shock protein or an active fragment thereof, the nucleic acid sequence being operatively linked to one or more transcription control sequences in a suitable expression system, enabling in vivo expression of the encoded protein or the active fragment thereof in a human host.  
     
     
         2 . The DNA vaccine of  claim 1 , wherein the mammalian heat shock protein is a human heat shock protein.  
     
     
         3 . The DNA vaccine of  claim 1 , wherein the heat shock protein is heat shock protein 60 (HSP60), heat shock protein 70 (HSP70) or heat shock protein 90 (HSP90).  
     
     
         4 . The DNA vaccine of  claim 3 , wherein the recombinant construct comprises a nucleic acid sequence encoding a fragment of HSP60, HSP70 or HSP90.  
     
     
         5 . The DNA vaccine of  claim 4 , wherein the nucleic acid sequence encodes a fragment of HSP60, with the fragment having an amino acid sequence selected from the group consisting of amino acids 1-140 of HSP60 (denoted as SEQ ID NO: 1), amino acids 130-260 of HSP60 (denoted as SEQ ID NO: 2), and amino acids 31-50 of HSP60 (denoted as SEQ ID NO: 3).  
     
     
         6 . The DNA vaccine of  claim 1 , wherein the T cell-mediated inflammatory autoimmune disease is rheumatoid arthritis, collagen II arthritis, multiple sclerosis, autoimmune neuritis, systemic lupus erythematosus, psoriasis, juvenile onset diabetes, Sjogren's disease, thyroid disease, sarcoidosis, autoimmune uveitis, inflammatory bowel disease (Crohn's and ulcerative colitis) or autoimmune hepatitis.  
     
     
         7 . The DNA vaccine of  claim 1 , wherein the transcription control sequences are selected from the group consisting of RSV control sequences, CMV control sequences, retroviral LTR sequences, SV-40 control sequences and (3-ACTIN? control sequences.  
     
     
         8 . The DNA vaccine of  claim 1 , wherein the recombinant construct is incorporated into an eukaryotic expression vector.  
     
     
         9 . The DNA vaccine of  claim 8 , wherein the eukaryotic expression vector is selected from the group consisting of PCDNA3, pcDNA3.1 (+/−), pZeoSV2 (+/−), pSecTag2, pDisplay, pEF/myc/cyto, PCMV/MYC/CYTO, pCR3.1, pCI, PBK-RSV, PBK-CMV and PTRES.  
     
     
         10 . A recombinant construct comprising a nucleic acid sequence encoding a fragment of mammalian HSP60, the fragment having an amino acid sequence selected from the group consisting of amino acids 1-140 of HSP60 (denoted as SEQ ID NO: 1), amino acids 130-260 of HSP60 (denoted as SEQ ID NO: 2), and amino acids 31-50 of HSP60 (denoted as SEQ ID NO: 3), with the nucleic acid sequence being operatively linked to one or more transcription control sequences.  
     
     
         11 . The construct of  claim 10 , wherein the mammalian HSP60 is human HSP60.  
     
     
         12 . The construct of  claim 10 , wherein the transcription control sequences are selected from the group consisting of RSV control sequences, CMV control sequences, retroviral LTR sequences, SV-40 control sequences and P-ACTIN control sequences.  
     
     
         13 . The construct of  claim 10 , wherein the recombinant construct is incorporated into an eukaryotic expression vector.  
     
     
         14 . The construct of  claim 13 , wherein the eukaryotic expression vector is selected from the group consisting of: PCDNA3, pcDNA3.1 (+/−), pZeoSV2 (+/−), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pCI, PBK-RSV, PBK-CMV and PTRES.  
     
     
         15 . A pharmaceutical composition for treating a T cell-mediated autoimmune disease comprising (a) a recombinant construct comprising an isolated nucleic acid sequence encoding a heat shock protein or an active fragment thereof, with the nucleic acid sequence being operatively linked to one or more transcription control sequences; and (b) a pharmaceutically acceptable carrier.  
     
     
         16 . The pharmaceutical composition of  claim 15 , which is useful for a T cell mediated inflammatory autoimmune disease other than insulin dependent diabetes mellitus (IDDM).  
     
     
         17 . The pharmaceutical composition of  claim 16 , wherein the T cell-mediated inflammatory autoimmune disease is rheumatoid arthritis, collagen II arthritis, multiple sclerosis, autoimmune neuritis, systemic lupus erythematosus, psoriasis, juvenile onset diabetes, Sjogren's disease, thyroid disease, sarcoidosis, autoimmune uveitis, inflammatory bowel disease (Crohn's and ulcerative colitis) or autoimmune hepatitis.  
     
     
         18 . The pharmaceutical composition of  claim 15 , wherein the heat shock protein is HSP60, HSP70 or HSP90, or an active fragment thereof.  
     
     
         19 . The pharmaceutical composition of  claim 18 , wherein the active fragment has an amino acid sequence selected from the group consisting of amino acids 1-140 of HSP60 (denoted as SEQ ID NO: 1), amino acids 130-260 of HSP60 (denoted as SEQ ID NO: 2), and amino acids 31-50 of HSP60 (denoted as SEQ ID NO: 3).  
     
     
         20 . The pharmaceutical composition of  claim 15 , wherein the carrier comprises a delivery vehicle that delivers the nucleic acid sequences to an individual.  
     
     
         21 . The pharmaceutical composition of  claim 20 , wherein the delivery vehicle is selected from the group consisting of liposomes, micelles, emulsions and cells.  
     
     
         22 . The pharmaceutical composition of  claim 15 , wherein the transcription control sequences are selected from the group consisting of RSV control sequences, CMV control sequences, retroviral LTR sequences, SV-40 control sequences and P-ACTIN control sequences.  
     
     
         23 . The pharmaceutical composition of  claim 15 , wherein the recombinant construct is incorporated into an eukaryotic expression vector.  
     
     
         24 . The pharmaceutical composition of  claim 23 , wherein the eukaryotic expression vector is selected from the group consisting of pcDNA3, pcDNA3.1 (+/−), pZeoSV2 (+/−), pSecTag2, pDisplay, pEF/myc/cyto, PCMV/MYC/CYTO, pCR3.1, pCI, PBK-RSV, PBK-CMV and PTRES.  
     
     
         25 . A method of treating a T cell-mediated inflammatory autoimmune disease, which comprises administering to an individual in need thereof a therapeutic composition comprising a recombinant construct that includes an isolated nucleic acid sequence encoding a heat shock protein or an active fragment thereof, with the nucleic acid sequence being operatively linked to one or more transcription control sequences, thereby treating the disease.  
     
     
         26 . The method of  claim 25 , wherein the heat shock protein is HSP60, HSP70, HSP90, or an active fragment thereof.  
     
     
         27 . The method of  claim 26 , wherein the active fragment having amino acid sequence selected from the group consisting of amino acids 1-140 of HSP60 (denoted as SEQ ID NO: 1), amino acids 130-260 of HSP60 (denoted as SEQ ID NO: 2), and amino acids 31-50 of HSP60 (denoted as SEQ ID NO: 3).  
     
     
         28 . The method of  claim 25 , wherein the T cell-mediated inflammatory autoimmune disease is rheumatoid arthritis, collagen II arthritis, multiple sclerosis, autoimmune neuritis, systemic lupus erythematosus, psoriasis, juvenile onset diabetes, Sjogren's disease, thyroid disease, sarcoidosis, autoimmune uveitis, inflammatory bowel disease (CROLM�S and ulcerative colitis) or autoimmune hepatitis.  
     
     
         29 . The method of  claim 25 , wherein the individual to be treated is selected from the group consisting of humans and non-human mammals.  
     
     
         30 . The method of  claim 25 , wherein the therapeutic composition is administered to the individual prior to the appearance of disease symptoms.  
     
     
         31 . The method of  claim 25 , wherein the therapeutic composition is administered by intravenous injection, intramuscular injection, or by aerosol, oral, percutaneous or topical administration.  
     
     
         32 . A method for treating a T cell-mediated inflammatory autoimmune disease which comprises the steps of (a) obtaining cells from an individual; (b) transfecting the cells in vitro with a recombinant construct comprising an isolated nucleic acid sequence encoding a heat shock protein or an active fragment thereof, said nucleic acid sequence being operatively linked to one or more transcription control sequences; and (c) reintroducing the transfected cells to the individual, thereby treating the disease.  
     
     
         33 . The method of  claim 32 , wherein the heat shock protein is HSP60, HSP70, HSP90, or an active fragment thereof.  
     
     
         34 . The method of  claim 33 , wherein the active fragment has an amino acid sequence selected from the group consisting of amino acids 1-140 of HSP60 (denoted as SEQ ID NO: 1), amino acids 130-260 of HSP60 (denoted as SEQ ID NO: 2), and amino acids 31-50 of HSP60 (denoted as SEQ ID NO: 3).  
     
     
         35 . The method of  claim 32 , wherein the T-cell mediated inflammatory autoimmune disease is rheumatoid arthritis, collagen II arthritis, multiple sclerosis, autoimmune neuritis, systemic lupus erythematosus, psoriasis, juvenile onset diabetes, Sjogren's disease, thyroid disease, sarcoidosis, autoimmune uveitis, inflammatory bowel disease (Crohn's and ulcerative colitis) or autoimmune hepatitis.  
     
     
         36 . The method of  claim 32 , wherein the individual to be treated is selected from the group consisting of humans and non-human mammals.  
     
     
         37 . A method of treating arthritis, which comprises administering to an individual in need thereof a therapeutic composition comprising (a) a fragment of mammalian HSP60 having amino acid sequence corresponding to amino acids 31-50 of HSP60 (denoted as SEQ ID NO: 3); and (b) a pharmaceutically acceptable carrier, thereby treating arthritis.  
     
     
         38 . The method of  claim 37 , wherein the carrier comprises a delivery vehicle that delivers the fragment to the individual.  
     
     
         39 . The method of  claim 38 , wherein the delivery vehicle is selected from the group consisting of liposomes, micelles, emulsions and cells.  
     
     
         40 . A method of treating arthritis which comprises the steps of (a) obtaining cells from an individual; (b) exposing the cells in vitro with an active amount of a fragment of mammalian HSP60 having amino acid sequence corresponding to amino acids 31-50 of HSP60 (denoted as SEQ ID NO: 3); and (c) reintroducing the exposed cells to the individual, thereby treating arthritis.  
     
     
         41 . The method of  claim 40 , wherein the cells are autologous T cells.  
     
     
         42 . The method of  claim 40 , wherein the mammalian HSP60 is human HSP60.