Patent EA-004330-B1 (EA)
Title: Hiv-1 tat, or derivatives thereof for prophylactic and therapeutic vaccination
Inventor: ENSOLI BARBARA
IPC: A61P35/00
Filed: 1998-11-30
Abstract: 1. Biologically active isolated Tat protein, having the sequence defined in SEQ. ID. NO 2, capable of: 1) entering activated endothelial cells or dendritic cells at concentrations up to 10 nM and 2) performing at least one action selected from the group consisting of the following actions: i) activating the proliferation, migration and invasion of Kaposi's sarcoma (KS) cells or cytokine-activated endothelial cells; ii) activating virus replication when added to infected cells as measured by a) the rescue of Tat-defective proviruses in HLM-1 cells after the addition of exogenous protein and/or b) the transactivation of HIV-1 gene expression in cells transfected with a HIV-1 promoter-reporter plasmid; and iii) inducing in mice the development of KS-like lesions in the presence of angiogenic factors or inflammatory cytokines. 2. Isolated DNA, defined in sequence SEQ. ID. NO 1, encoding Tat protein according to claim 1, which is capable of: 1) entering activated endothelial cells or dendritic cells at concentrations up to 10 nM and 2) performing at least one action selected from the group consisting of the following actions: i) activating the proliferation, migration and invasion of Kaposi's sarcoma (KS) cells or cytokine-activated endothelial cells; ii) activating virus replication when added to infected cells as measured by a) the rescue of Tat-defective proviruses in HLM-1 cells after the addition of exogenous protein and/or b) the transactivation of HIV-1 gene expression in cells transfected with a HIV-1 promoter-reporter plasmid; and iii) inducing in mice the development of KS-like lesions in the presence of angiogenic factors or inflammatory cytokines. 3. Biologically active isolated fragment of Tat protein according to claim 1, having a sequence, selected from the group of the following sequences: SEQ. ID. N 11, SEQ. ID. N 12, SEQ. ID. N 13, SEQ. ID. N 14, SEQ. ID. N 15, SEQ. ID. N 16, SEQ. ID. N 17, and capable of: 1) entering activated endothelial cells or dendritic cells at concentrations up to 10 nM and 2) performing at least one action selected from the group consisting of the following actions: i) activating the proliferation, migration and invasion of Kaposi's sarcoma (KS) cells or cytokine-activated endothelial cells; ii) activating virus replication when added to infected cells as measured by a) the rescue of Tat-defective proviruses in HLM-1 cells after the addition of exogenous protein and/or b) the transactivation of HIV-1 gene expression in cells transfected with a HIV-1 promoter-reporter plasmid; and iii) inducing in mice the development of KS-like lesions in the presence of angiogenic factors or inflammatory cytokines. 4. Biologically active isolated mutant of Tat protein, according to claim 1, having a sequence selected from group sequences: amino acid sequence of cys22, identified as SEQ. ID. NO 7; amino acid sequence of lys41, identified as SEQ. ID. NO 8; amino acid sequence of RGDDelta, identified as SEQ. ID. NO 9; amino acid sequence of lys41-RGDDelta mutant, identified as SEQ. ID. NO 10, capable of: 1) entering activated endothelial cells or dendritic cells at concentrations up to 10 nM and 2) performing at least one action selected from the group consisting of the following actions: i) activating the proliferation, migration and invasion of Kaposi's sarcoma (KS) cells or cytokine-activated endothelial cells; ii) activating virus replication when added to infected cells as measured by a) the rescue of Tat-defective proviruses in HLM-1 cells after the addition of exogenous protein and/or b) the transactivation of HIV-1 gene expression in cells transfected with a HIV-1 promoter-reporter plasmid; and iii) inducing in mice the development of KS-like lesions in the presence of angiogenic factors or inflammatory cytokines. 5. Isolated DNA, encoding biologically active mutant of Tat protein, which is capable of: 1) entering activated endothelial cells or dendritic cells at concentrations up to 10 nM and 2) performing at least one action selected from the group consisting of the following actions: i) activating the proliferation, migration and invasion of Kaposi's sarcoma (KS) cells or cytokine-activated endothelial cells; ii) activating virus replication when added to infected cells as measured by a) the rescue of Tat-defective proviruses in HLM-1 cells after the addition of exogenous protein and/or b) the transactivation of HIV-1 gene expression in cells transfected with a HIV-1 promoter-reporter plasmid; and iii) inducing in mice the development of KS-like lesions in the presence of angiogenic factors or inflammatory cytokines, wherein said sequence is a sequence representing nucleotide sequence of cys22 mutant, identified as SEQ. ID. NO 3; nucleotide sequence of lys41, identified as SEQ. ID. NO 4; nucleotide sequence of RGDDelta mutant, identified as SEQ. ID. NO 5; nucleotide sequence of lys41-RGDDelta mutant, identified as SEQ. ID. NO 6. 6. Biologically active isolated Tat protein, fragments thereof and/or mutants and/or Tat DNA according to claims 1-5, useful in the prophylactic and/or therapeutic treatment of AIDS, tumors, syndromes and symptoms associated with HIV infection. 7. Prophylactic and/or medical vaccine effective against AIDS tumors, syndromes and symptoms associated with HIV infection on the base of biologically active isolated protein Tat, fragments and/or mutant thereof, or DNA Tat according to claim 1-5. 8. A pharmaceutical composition comprising as active principle biologically active isolated Tat protein, fragments thereof and/or mutants and/or Tat DNA according to claims 1-5 in combination with suitable excipients and/or diluents (for use in the prophylactic and/or therapeutic treatment of AIDS, tumors, syndromes and symptoms associated with HIV infection). 9. A pharmaceutical composition according to claim 8 for use in the prophylactic and/or therapeutic treatment of AIDS, tumors, syndromes and symptoms associated with HIV infection. 10. The pharmaceutical composition according to claim 8, wherein said composition is a vaccine for use in the prophylactic and/or therapeutic treatment of AIDS, tumors, syndromes and symptoms associated with HIV infection. 11. The pharmaceutical composition according to claim 8 wherein Tat, in its purified, non-aggregated and non-oxidized form, is lyophilized for storage and re-suspended in a biologically acceptable fluid for use. 12. The pharmaceutical composition according to any one of claims 8-11 in a form suitable for administration selected from the group consisting of mucosal, nasal, oral, vaginal, rectal, intramuscular, subcutaneous, intradermal, systemic and local administration. 13. A pharmaceutical composition comprising, as active principle biologically active isolated Tat protein, fragments thereof and/or mutants and/or Tat DNA in combination with suitable excipients and/or diluents, wherein said Tat is obtained by a process that includes a purification step under conditions to prevent Tat protein oxidation and aggregation. 14. The pharmaceutical composition according to claim 13, wherein said purification step is performed by heparin affinity chromatography. 15. The pharmaceutical composition according to claim 13, wherein said purification step is followed by storage of the Tat protein in lyophilized form and its re-suspension in a degassed buffer. 16.The pharmaceutical composition according to claim 13, wherein said composition is a vaccine for use in the prophylactic and/or therapeutic treatment of AIDS, tumors, syndromes and symptoms associated with HIV infection. 17.The pharmaceutical composition according to claim 13, wherein Tat has the sequence identified as SEQ. ID. NO 1. 18.The pharmaceutical composition according to claim 13, wherein Tat has the sequence identified as SEQ. ID. NO 2. 19.The pharmaceutical composition according to any one of claims 13-16 wherein the mutants are selected in the group of the ones having the following nucleotide sequences or part of them: Nucleotide sequence of cys22 mutant, identified as SEQ. ID. NO 3; Nucleotide sequence of lys41, identified as SEQ. ID. NO 4; Nucleotide sequence of RGDDelta mutant, identified as SEQ. ID. NO 5; Nucleotide sequence of lys41-RGDDelta mutant, identified as SEQ. ID. NO 6. 20.The pharmaceutical composition according to claims 13-16 wherein the mutants are selected in the group of the ones having the following amino acid sequences or part of them: Amino acid sequence of cys22 mutant, identified as SEQ. ID. NO 7; Amino acid sequence of lys41, identified as SEQ. ID. NO 8; Amino acid sequence of RGDDelta mutant, identified as SEQ. ID. NO 9; Amino acid sequence of lys41- RGDDelta mutant, identified as SEQ. ID. NO 10. 21. The pharmaceutical composition according to any one of claims 13-16 wherein said Tat fragment is selected from the group of peptides having sequences, identified as SEQ. ID. NO 11, SEQ.ID. NO 12, SEQ. ID. NO 13, SEQ. ID. NO 14, SEQ. ID. NO 15, SEQ. ID. NO16andSEQ. ID. NO 17. 22. The pharmaceutical composition according to any one of claims 13-21 in a form suitable for administration selected from the group consisting of mucosal, nasal, oral, vaginal, rectal, intramuscular, subcutaneous, intradermal, systemic and local administration. 23. The pharmaceutical composition according to any one of claims 8-22 comprising proteins or peptides conjugated with the T-helper universal epitope of Tetanus toxoid or any T-helper peptides. 24. The pharmaceutical composition according to claims 8-22 in combination with recombinant proteins or peptides of HIV Nef, Rev or Gag or part of them. 25. The pharmaceutical composition according to any one of claims 8-22 comprising fusion proteins Tat (wild type or its mutants)/Nef, Tat (wild type or its mutants)/Rev, Tat (wild type or its mutants)/Gag or part of them. 26. The pharmaceutical composition according to claims 9-27 in combination with recombinant immuno-modulant cytokines or other molecules, or part of them,