# US 7,744,896 B1 · HIV-1 Tat compositions > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 7,744,896 B1 - Document ID: US7744896B1 - Status: Granted - Kind: B1 - Application number: 09/555,534 - Filed: 1998-11-30 - Granted: 2010-06-29 - Earliest claimed: 1997-12-01 - Assignees: Istituto Superiore di Sanita - Inventors: Barbara Ensoli - Web page: https://patentgrep.com/patent/US7744896B1 ## CPC - A61K 39/21: Retroviridae, e.g. equine infectious anemia virus - A61K 39/12: Viral antigens - A61K 40/19: Dendritic cells - A61K 40/24: Antigen-presenting cells [APC] - A61K 40/46: Viral antigens - A61P 31/12: Antivirals - A61P 31/18: for HIV - A61P 35/00: Antineoplastic agents - A61P 37/04: Immunostimulants - C07K 14/005: from viruses - A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) - A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53) - A61K 2039/525: {Virus} - A61K 2039/53: {DNA (RNA) vaccination} - A61K 2039/545: {characterised by the dose, timing or administration schedule} - A61K 2039/55505: {Inorganic adjuvants} - A61K 2039/55566: {Emulsions, e.g. Freund's adjuvant, MF59} - A61K 2239/31: characterized by the route of administration - A61K 2239/38: characterised by the dose, timing or administration schedule - C12N 2710/16043: viral genome or elements thereof as genetic vector - C12N 2740/16043: viral genome or elements thereof as genetic vector - C12N 2740/16322: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes - C12N 2740/16334: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The present invention refers to Tat as the active principle for a prophylactic and/or therapeutic vaccine against HIV infection, the progression towards AIDS and the development of tumors and other syndromes and symptoms in subjects infected by HIV. Tat is in biologically active form either as recombinant protein or peptide or as DNA. More particularly, the invention refers to a vaccine based on HIV-1 Tat as immunogen, inoculated as DNA and/or recombinant protein or as peptides, alone or in combination with other genes or viral gene products (Nef, Rev, Gag) or parts thereof, or in combination with various immuno modulant cytokines (IL-12, IL-15) or with the gene coding for an immuno modulant cytokine or part thereof. Tat, Nef, Rev, Gag and the immuno modulant cytokines are administrated both as a mixture of recombinant proteins, peptides or fusion proteins (Tat/Nef, Tat/Rev, Tat/Gag, Tat/IL-12, Tat/IL-15) or as plasmid DNA. ## Claims ### Claim 1 A composition comprising: an isolated HIV Tat protein in combination with a pharmaceutically acceptable carrier or excipient, wherein said isolated HIV Tat protein is biologically active, as shown by (1) the ability of said isolated HIV Tat protein to activate virus replication when said isolated HIV Tat protein is added to HIV-1 infected cells, which ability to activate is determined by (A) the rescue of Tat-defective proviruses in HLM-1 cells after the addition of said isolated HIV Tat protein at a concentration of up to 1 μg/ml, or (B) the transactivation of HIV-1 gene expression in cells transfected with HIV-1 promoter-reporter plasmid after the addition of said isolated HIV Tat protein at a concentration of up to 1 μg/ml; and (2) the ability of said isolated HIV Tat protein to do one or both of the following (i) and (ii): (i) enter and localize in the nuclei of activated endothelial cells or dendritic cells, which entering and localizing is determined by (a) incubating activated endothelial cells or dendritic cells with up to 1 μg/ml of said isolated HIV Tat protein which is labeled with rhodamine, and (b) detecting the presence or absence of rhodamine in the activated endothelial cells or dendritic cells by fluorescence microscopy; or (ii) activate the proliferation, migration, and invasion of Kaposi's sarcoma (KS) cells or cytokine-activated endothelial cells in culture when said isolated HIV Tat protein is present at a concentration of up to 1 μg/ml, wherein said composition is pharmaceutically acceptable for administration to a human. ### Claim 2 (depends on 1) The composition of claim 1, wherein said isolated HIV Tat protein is purified. ### Claim 3 (depends on 1) The composition of claim 1, wherein said isolated HIV Tat protein is a wild type HIV Tat protein. ### Claim 4 (depends on 1, 2, 3) The composition of claim 1, 2 or 3, wherein the administration is selected from the group consisting of mucosal, nasal, oral, vaginal, rectal, intramuscular, subcutaneous, intradermal, systemic, and local administration. ### Claim 5 (depends on 2) The composition of claim 2, wherein said isolated HIV Tat protein is purified by a method comprising performing heparin affinity chromatography. ### Claim 6 (depends on 5) The composition of claim 5, wherein said performing step is followed by steps of (a) lyophilizing said isolated HIV Tat protein, and (b) resuspending said lyophilized isolated HIV Tat protein in a degassed buffer. ### Claim 7 (depends on 1, 2, 3) The composition of claim 1, 2 or 3 which further comprises a biologically acceptable fluid. ### Claim 8 (depends on 1, 2, 3) A product which is produced by a process comprising lyophilizing the composition of claim 1, 2 or 3. ### Claim 9 (depends on 1, 2, 3) A product which is produced by a process comprising lyophilizing the composition of claim 1, 2 or 3 and resuspending the lyophilized composition in a biologically acceptable fluid. ### Claim 10 (depends on 3) The composition of claim 3, wherein the amino acid sequence of said wild type HIV Tat protein consists of SEQ ID NO:2. ### Claim 11 (depends on 7) The composition of claim 7, wherein the biologically acceptable fluid is serum, plasma, or one or more fractions thereof. ### Claim 12 (depends on 9) The product of claim 9, wherein the biologically acceptable fluid is serum, plasma, or one or more fractions thereof. ### Claim 13 (depends on 1, 2, 3) The composition of claim 1, 2, 3, or 6 which further comprises an adjuvant. ### Claim 14 (depends on 13) The composition of claim 13 which further comprises a biologically acceptable fluid. ### Claim 15 (depends on 13) The composition of claim 13, wherein the adjuvant is RIBI, alum, or ISCOM, or a combination thereof. ### Claim 16 (depends on 1, 2, 3) The composition of claim 1, 2 or 3, wherein said isolated HIV Tat protein is bound to a delivery vehicle. ### Claim 17 (depends on 16) The composition of claim 16, wherein said delivery vehicle is a nanoparticle. ### Claim 18 (depends on 16) The composition of claim 16, wherein said delivery vehicle is an autologous erythrocyte. ### Claim 19 (depends on 4) The composition of claim 4, wherein the administration is systemic. ### Claim 20 (depends on 4) The composition of claim 4, wherein the administration is intradermal. ### Claim 21 (depends on 4) The composition of claim 4, wherein the administration is subcutaneous. ### Claim 22 (depends on 4) The composition of claim 4, wherein the administration is mucosal. ### Claim 23 (depends on 13) The composition of claim 13, wherein the administration is selected from the group consisting of mucosal, nasal, oral, vaginal, rectal, intramuscular, subcutaneous, intradermal, systemic, and local administration. ### Claim 24 (depends on 23) The composition of claim 23, wherein the administration is systemic. ### Claim 25 (depends on 23) The composition of claim 23, wherein the administration is intradermal. ### Claim 26 (depends on 23) The composition of claim 23, wherein the administration is subcutaneous. ### Claim 27 (depends on 26) The composition of claim 26 which further comprises Alum. ### Claim 28 (depends on 23) The composition of claim 23, wherein the administration is mucosal. ### Claim 29 (depends on 1, 2, 3) The composition of claim 1, 2, 3, or 4, wherein said isolated HIV Tat protein is conjugated to a T-helper peptide or T-helper universal epitope of Tetanus Toxoid. ### Claim 30 (depends on 1, 2, 3, 6) The composition of claim 1, 2, 3 or 6, which further comprises HIV Rev, Nef or Gag, or an immunogenic fragment thereof. ### Claim 31 (depends on 1, 2, 3) The composition of claim 1, 2, 3, or 4, which further comprises an immuno-modulant cytokine. ### Claim 32 (depends on 31) The composition of claim 31, wherein said immuno-modulant cytokine is IL-12 or IL-15. ### Claim 33 (depends on 1, 2, 3, 6) The composition of claim 1, 2, 3 or 6, wherein said isolated HIV Tat protein is fused to HIV Rev, Nef or Gag, or an immunogenic fragment thereof. ### Claim 34 (depends on 1, 2, 3) The composition of claim 1, 2, 3, or 4, wherein said isolated HIV Tat protein is fused to an immuno-modulant cytokine. ### Claim 35 (depends on 34) The composition of claim 34, wherein said immuno-modulant cytokine is IL-12 or IL-15. ### Claim 36 (depends on 1, 2, 3) The composition of claim 1, 2, 3, or 6, which further comprises an inhibitor of viral replication. ### Claim 37 (depends on 1) The composition of claim 1 which comprises a combination of said isolated HIV Tat protein. ### Claim 38 (depends on 1, 2) The composition of claim 1 or 2 which is suitable for inducing an immune response in the human to said isolated HIV Tat protein. ### Claim 39 (depends on 3) The composition of claim 3, wherein said wild type HIV Tat protein is purified. ### Claim 40 (depends on 10) The composition of claim 10, wherein said wild type HIV Tat protein is purified. ### Claim 41 (depends on 1) The composition of claim 1, wherein said isolated HIV Tat protein is biologically active, as shown by (1) the ability of said isolated HIV Tat protein to activate virus replication when said isolated HIV Tat protein is added to HIV-1 infected cells, and (2) the ability of said isolated HIV Tat protein to enter and localize in the nuclei of activated endothelial cells or dendritic cells; which ability to activate is determined by (A) the rescue of Tat-defective proviruses in HLM-1 cells after the addition of said isolated HIV Tat protein at a concentration of up to 1 μg/ml, or (B) the transactivation of HIV-1 gene expression in cells transfected with a HIV-1 promoter-reporter plasmid after the addition of said isolated HIV Tat protein at a concentration of up to 1 μg/ml, and which ability to enter and localize is determined by (a) incubating activated endothelial cells or dendritic cells with up to 1 μg/ml of said isolated HIV Tat protein which is labeled with rhodamine, and (b) detecting the presence or absence of rhodamine in the activated endothelial cells or dendritic cells by fluorescence microscopy. ### Claim 42 (depends on 41) The composition of claim 41, wherein the isolated HIV Tat protein is a wild type HIV Tat protein. ### Claim 43 (depends on 42) The composition of claim 42, wherein said wild type HIV Tat protein is purified. ### Claim 44 (depends on 42) The composition of claim 42, wherein the amino acid sequence of said wild type HIV Tat protein consists of SEQ ID NO:2. ### Claim 45 (depends on 44) The composition of claim 44, wherein said wild type HIV Tat protein is purified. ### Claim 46 (depends on 41, 42, 43) The composition of claim 41, 42, 43, or 45 which further comprises an adjuvant. ### Claim 47 (depends on 41, 42, 43) The composition of claim 41, 42, 43, or 45, wherein the administration is intradermal. ### Claim 48 (depends on 41, 42, 43) The composition of claim 41, 42, 43, or 45, wherein the administration is subcutaneous. ### Claim 49 (depends on 48) The composition of claim 48 which further comprises Alum. ### Claim 50 (depends on 1) The composition of claim 1, wherein said isolated HIV Tat protein is biologically active, as shown by (1) the ability of said isolated HIV Tat protein to activate virus replication when said isolated HIV Tat protein is added to HIV-1 infected cells, which ability to activate is determined by (A) the rescue of Tat-defective proviruses in HLM-1 cells after the addition of said isolated HIV Tat protein at a concentration of up to 1 μg/ml, or (B) the transactivation of HIV-1 gene expression in cells transfected with a HIV-1 promoter-reporter plasmid after the addition of said isolated HIV Tat protein at a concentration of up to 1 μg/ml; and (2) the ability of said isolated HIV Tat protein to do both of the following (i) and (ii): (i) enter and localize in the nuclei of activated endothelial cells or dendritic cells, which entering and localizing is determined by (a) incubating activated endothelial cells or dendritic cells with up to 1 μg/ml of said isolated HIV Tat protein which is labeled with rhodamine, and (b) detecting the presence or absence of rhodamine in the activated endothelial cells or dendritic cells by fluorescence microscopy; and (ii) activate the proliferation, migration, and invasion of Kaposi's sarcoma (KS) cells or cytokine-activated endothelial cells in culture when said isolated HIV Tat protein is present at a concentration of up to 1 μg/ml. ### Claim 51 (depends on 50) The composition of claim 50, wherein the isolated HIV Tat protein is a wild type HIV Tat protein. ### Claim 52 (depends on 51) The composition of claim 51, wherein said wild type HIV Tat protein is purified. ### Claim 53 (depends on 51) The composition of claim 51, wherein the amino acid sequence of said wild type HIV Tat protein consists of SEQ ID NO:2. ### Claim 54 (depends on 53) The composition of claim 53, wherein said wild type HIV Tat protein is purified. ### Claim 55 (depends on 50, 51, 52) The composition of claim 50, 51, 52, or 54 which further comprises an adjuvant. ### Claim 56 (depends on 50, 51, 52) The composition of claim 50, 51, 52, or 54, wherein the administration is intradermal. ### Claim 57 (depends on 50, 51, 52) The composition of claim 50, 51, 52, or 54, wherein the administration is subcutaneous. ### Claim 58 (depends on 57) The composition of claim 57 which further comprises Alum. ### Claim 59 (depends on 1, 2) The composition of claim 1 or 2, wherein said isolated HIV Tat protein comprises the cysteine rich region of HIV Tat and is in a non-oxidated form. ### Claim 60 (depends on 10) The composition of claim 10, wherein said wild type HIV Tat protein is in a non-oxidated form. ### Claim 61 (depends on 42, 43, 44, 45) The composition of any of claims 42-45, wherein said wild type HIV Tat protein is in a non-oxidated form. ### Claim 62 (depends on 51, 52, 53, 54) The composition of any of claims 51-54, wherein said wild type HIV Tat protein is in a non-oxidated form. ### Claim 63 A composition comprising: an isolated HIV Tat protein in combination with a pharmaceutically acceptable carrier or excipient, wherein said isolated HIV Tat protein is in a non-oxidated form, wherein said composition is pharmaceutically acceptable for administration to a human, and wherein the isolated HIV Tat protein is a wild type HIV Tat protein. ### Claim 64 (depends on 63) The composition of claim 63, wherein said wild type HIV Tat protein is purified. ### Claim 65 (depends on 63) The composition of claim 63, wherein the amino acid sequence of said wild type HIV Tat protein consists of SEQ ID NO:2. ### Claim 66 (depends on 65) The composition of claim 65, wherein said wild type HIV Tat protein is purified. ### Claim 67 (depends on 63, 64) The composition of claim 63, 64, or 66 which further comprises an adjuvant. ### Claim 68 (depends on 63, 64) The composition of claim 63, 64, or 66, wherein the administration is intradermal. ### Claim 69 (depends on 63, 64) The composition of claim 63, 64, or 66, wherein the administration is subcutaneous. ### Claim 70 (depends on 69) The composition of claim 69 which further comprises Alum. ### Claim 71 (depends on 63) The composition of claim 63, wherein said isolated HIV Tat protein is conjugated to a T-helper peptide or T-helper universal epitope of Tetanus Toxoid. ### Claim 72 (depends on 63) The composition of claim 63, which further comprises HIV Rev, Nef or Gag, or an immunogenic fragment thereof. ### Claim 73 (depends on 63) The composition of claim 63, which further comprises an immuno-modulant cytokine. ### Claim 74 (depends on 73) The composition of claim 73, wherein said immuno-modulant cytokine is IL-12 or IL-15. ### Claim 75 (depends on 1) The composition of claim 1, wherein said isolated HIV Tat protein is biologically active, as shown by (1) the ability of said isolated HIV Tat protein to activate virus replication when said isolated HIV Tat protein is added to HIV-1 infected cells, and (2) the ability of said isolated HIV Tat protein to activate the proliferation, migration, and invasion of Kaposi's sarcoma (KS) cells or cytokine-activated endothelial cells in culture when said isolated HIV Tat protein is present at a concentration of up to 1 μg/ml; which ability to activate is determined by (A) the rescue of Tat-defective proviruses in HLM-1 cells after the addition of said isolated HIV Tat protein at a concentration of up to 1 μg/ml, or (B) the transactivation of HIV-1 gene expression in cells transfected with a HIV-1 promoter-reporter plasmid after the addition of said isolated HIV Tat protein at a concentration of up to 1 μg/ml. ### Claim 76 (depends on 75) The composition of claim 75, wherein the isolated HIV Tat protein is a wild type HIV Tat protein. ### Claim 77 (depends on 76) The composition of claim 76, wherein said wild type HIV Tat protein is purified. ### Claim 78 (depends on 76) The composition of claim 76, wherein the amino acid sequence of said wild type HIV Tat protein consists of SEQ ID NO:2. ### Claim 79 (depends on 78) The composition of claim 78, wherein said wild type HIV Tat protein is purified. ### Claim 80 (depends on 75, 76, 77) The composition of claim 75, 76, 77, or 79 which further comprises an adjuvant. ### Claim 81 (depends on 75, 76, 77) The composition of claim 75, 76, 77, or 79, wherein the administration is intradermal. ### Claim 82 (depends on 75, 76, 77) The composition of claim 75, 76, 77, or 79, wherein the administration is subcutaneous. ### Claim 83 (depends on 82) The composition of claim 82 which further comprises Alum.