WO2009026183A1 - Utilisation de protéines gag vih/vis chimériques pour optimiser des réponses de lymphocytes t induites par vaccin contre le gag du vih - Google Patents

Utilisation de protéines gag vih/vis chimériques pour optimiser des réponses de lymphocytes t induites par vaccin contre le gag du vih Download PDF

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WO2009026183A1
WO2009026183A1 PCT/US2008/073395 US2008073395W WO2009026183A1 WO 2009026183 A1 WO2009026183 A1 WO 2009026183A1 US 2008073395 W US2008073395 W US 2008073395W WO 2009026183 A1 WO2009026183 A1 WO 2009026183A1
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hiv
gag
amino acids
vector
nucleic acid
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PCT/US2008/073395
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Gary J. Nabel
Zhi-Yong Yang
Wei Shi
Dan H. Barouch
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The Government Of The U.S.A., As Represented By The Secretary, Department Of Health & Human Services
Beth Israel Deaconess Medical Center
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Publication of WO2009026183A1 publication Critical patent/WO2009026183A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • Modified HIV-I Gag polypeptides in which one or more amino acids in the HIV-I Gag polypeptide have been replaced with one or more amino acids from the corresponding region in the SIV Gag protein and nucleic acids encoding the modified HIV-I Gag polypeptides are disclosed. Methods and compositions for using the nucleic acids and modified HIV-I Gag polypeptides to induce an immune response are also disclosed.
  • AIDS is the fifth leading cause of death among persons between the ages of 25 and 44 in the United States. About 47 million people worldwide have been infected with HIV-I since the start of the epidemic. HIV-I infections continue to rise due to the increase of drug-resistant strains of the disease. Many of the existing drugs used for treating HIV have undesirable side effects [0005] Accordingly, it is desirable to develop new approaches to reducing the incidence of HIV-I infection or ameliorating the consequences of HIV-I infection. Vaccines are particularly desirable in this regard.
  • One embodiment disclosed herein is a nucleic acid encoding an HIV-I Gag polypeptide comprising a modified amino acid sequence comprising an amino acid sequence in which one or more amino acids in said modified HIV-I Gag polypeptide have been replaced with one or more amino acids from the corresponding region in the SIV Gag protein.
  • themodified amino acid sequence comprises a sequence in which at least a portion of the 179 carboxy terminal amino acids of the HIV-I Gag polypeptide has been replaced with an amino acid sequence comprising the corresponding portion of the SIV Gag protein.
  • the modified amino acid sequence comprises a sequence in which said at least a portion of the 179 carboxy terminal amino acids of the HIV-I Gag polypeptide is at least about 5 contiguous amino acids in length, at least about 10 contiguous amino acids in length, at least about 20 contiguous amino acids in length, at least about 30 contiguous amino acids in length, at least about 40 contiguous amino acids in length, at least about 50 contiguous amino acids in length, at least about 60 contiguous amino acids in length or more than 60 contiguous amino acids in length.
  • the modified amino acid sequence comprises a sequence in which amino acids 324-500 of the HIV-I Gag polypeptide have been replaced with amino acids 333-510 from the SIV Gag protein.
  • the modified amino acid sequence comprises a sequence in which at least a portion of the amino acid sequence between amino acids 358-411 of the HIV-I Gag polypeptide has been replaced with at least a portion of the corresponding region of the SIV Gag protein.
  • the modified amino acid sequence comprises a sequence in which amino acids 358-41 1 of the HIV-I Gag polypeptide have been replaced with amino acids 359-412 of the SIV Gag protein.
  • the modified amino acid sequence comprises a sequence in which at least a portion of the amino acid sequence between amino acids 419-454 of the HIV-I Gag polypeptide has been replaced with at least a portion of the corresponding region of the SIV Gag protein.
  • the modified amino acid sequence comprises a sequence in which amino acids 419-454 of the HIV-I Gag polypeptide have been replaced with amino acids 420-457 of the SIV Gag protein. In still further aspects of this embodiment, the modified amino acid sequence comprises a sequence in which at least a portion of the amino acid sequence between amino acids 214-225 or 332-500 of the HIV-I Gag polypeptide have been replaced with at least a portion of the corresponding region of the SIV Gag protein.
  • the modified amino acid sequence comprises a sequence in which amino acids 214-225 of the HIV-I Gag polypeptide have been replaced with amino acids 216-225 of the SIV Gag protein and amino acids 332-500 of the HIV-I Gag polypeptide have been replaced with amino acids 333-510 of the SIV Gag protein.
  • the modified amino acid sequence comprises a sequence in which at least a portion of the amino acid sequence between amino acids 214- 254 or 332-500 of the HIV-I Gag polypeptide have been replaced with at least a portion of the corresponding region of the SIV Gag protein.
  • the modified amino acid sequence comprises a sequence in which amino acids 214-254 of the HIV-I Gag polypeptide have been replaced with amino acids 216-255 of the SIV Gag protein and amino acids 332-510 of the HIV-I Gag polypeptide have been replaced with amino acids 333-510 of the SIV Gag protein.
  • at least a portion of the HIV-I Gag protein of SEQ ID NO: 2 has been replaced with at least a portion of the SIV Gag protein of SEQ ID NO: 1.
  • the HIV-I Gag polypeptide is a fragment of the HIV-I protein and wherein a portion of said fragment has been replaced with one or more amino acids from the corresponding region of the SIV Gag protein.
  • the HIV-I Gag polypeptide comprises one or more antigenic regions capable of inducing an immune response against HIV-I .
  • Another embodiment disclosed herein is a nucleic acid encoding an HIV-I Gag polypeptide comprising a plurality of the modified amino acid sequences recited in the preceding paragraph.
  • the vector is a viral vector.
  • the viral vector is an adenovirus vector.
  • the adenovirus vector is an adenovirus 5 vector.
  • the adenovirus vector is an adenovirus 26 vector.
  • the vector is a gene-based vector.
  • the vector is a plasmid DNA vector used alone or in combination with an adjuvant.
  • Another embodiment disclosed herein is a host cell comprising any of the vectors in the preceding paragraph, wherein the host cell is suitable for expressing said HIV-I Gag polypeptide.
  • compositions capable of eliciting an immune response against HIV-I comprising any of the nucleic acids or vectors described above and a pharmaceutically acceptable carrier.
  • Another embodiment disclosed herein is a syringe or a needless delivery device comprising the composition of the preceding paragraph.
  • nucleic acid comprises any of the vectors described above.
  • Another embodiment disclosed herein is a method of inducing an immune response against HIV-I comprising administering any of the polypeptides described above to an individual.
  • Another embodiment disclosed herein is the use of any of the nucleic acids or vectors disclosed above to induce an immune response in an individual.
  • Another embodiment disclosed herein is the use of any of the polypeptides described above to induce an immune response in an individual.
  • Figure 1 shows an alignment of the sequence of the SIV Gag protein (SEQ ID NO: 1) with the sequence of the HIV-I Gag protein (SEQ ID NO: 2).
  • Figure 2 shows the results of a Western Blot demonstrating the expression of modified HIV-I Gag polypeptides.
  • Figure 3 shows the structure of the CMVR gag #1 plasmid (SEQ ID NO: 3).
  • Figure 4 shows the structure of the CMVR gag 2 plasmid (SEQ ID NO: 5).
  • Figure 5 shows the structure of the CMVR gag 3 plasmid (SEQ ID NO: 7).
  • Figure 6 shows the structure of the CMVR gag 4 plasmid (SEQ ID NO: 9).
  • Figure 7 shows the structure of the CMVR gag 5 plasmid (SEQ ID NO: 11).
  • Figure 8 shows the structure of the CMVR gag 6 plasmid (SEQ ID NO: 13).
  • Figure 9 shows the structure of the CMVR SIV-gag-HIV-pol #7 plasmid (SEQ ID NO: 15).
  • Figure 10 shows the structure of the CMVR SIV gag #8 plasmid (SEQ ID NO: 17).
  • Figure 1 1 shows the structure of the CMVR gag 9 plasmid (SEQ ID NO: 19).
  • Figure 12 shows the structure of the CMVR gag 10 plasmid (SEQ ID NO: 21).
  • Figure 13 shows the structure of the CMVR gag 11 plasmid (SEQ ID NO: 23).
  • Figure 14 shows the structure of the CMVR gag 12 plasmid (SEQ ID NO: 25).
  • Figure 15 shows the structure of the CMVR gag 13 plasmid (SEQ ID O: 27).
  • Figure 16 shows the structure of the CMVR gag 14 plasmid (SEQ ID O: 29).
  • Figure 17 shows the structure of the CMVR gag new 2 plasmid (SEQ ID NO: 31).
  • Figure 18 shows the structure of the CMVR gag new 3 plasmid (SEQ ID NO: 33).
  • Figure 19 shows the structure of the CMVR gag new 4 plasmid (SEQ ID NO: 35).
  • Figure 20 shows the structure of the CMVR gag new 5 plasmid (SEQ ID NO: 37).
  • Figure 21 shows the structure of the CMVR gag new 6 plasmid (SEQ ID NO: 39).
  • Figure 22 shows the structure of the CMVR gag new 7 plasmid (SEQ ID NO: 41).
  • Figure 23 shows the structure of the CMVR gag new 8 plasmid (SEQ ID NO: 43).
  • Figure 24 shows the structure of the CMVR gag new 9 plasmid (SEQ ID NO: 45).
  • Figure 25 shows the structure of the CMVR gag new 10 plasmid (SEQ ID NO: 47).
  • Figure 26 shows the structure of the CMVR gag new 1 1 plasmid (SEQ ID NO: 49).
  • Figure 27 shows the structure of the CMVR gag new 14 plasmid (SEQ ID NO: 51).
  • Figure 28 shows the results of an immunogenicity assay with several plasmids expressing modified HIV-I Gag polypeptides.
  • Figure 29 shows the results of an immunogenicity assay with several plasmids expressing modified HIV-I Gag polypeptides.
  • Figure 30 shows the results of an immunogenicity assay with several plasmids expressing modified HIV-I Gag polypeptides
  • Figure 31 shows the structure of the CMVR gag new 1 plasmid (SEQ ID NO: 53).
  • Figure 32 shows the structure of the CMVR gag new 4-1 plasmid (SEQ ID NO: 55).
  • Figure 33 shows the structure of the CMVR gag new 5-1 plasmid (SEQ ID NO: 57).
  • Figure 34 shows the structure of the CMVR gag new 6-1 plasmid (SEQ ID NO: 59).
  • Figure 35 shows the structure of the CMVR gag new Pl plasmid (SEQ ID NO: 61).
  • Figure 36 shows the structure of the CMVR gag new PlB plasmid (SEQ ID NO: 63).
  • Figure 37 shows the structure of the CMVR gag new 12 plasmid (SEQ ID NO: 65).
  • Figure 38 shows the structure of the CMVR gag new 13 plasmid (SEQ ID NO: 67).
  • Figure 39 shows the structure of the CMVR gag new 15 plasmid (SEQ ID NO: 69).
  • Figure 40 shows the structure of the CMVR gag new 16 plasmid (SEQ ID NO: 71).
  • Figure 41 shows the structure of the CMVR gag new 17 plasmid (SEQ ID NO: 73).
  • Figure 42 is a graphical representation of CMVR gag new 1 , CMVR gag new 4-1, CMVR gag new 5-1, CMVR gag new 6-1, CMVR gag new Pl, and CMVR gag new PlB.
  • Figure 43 shows additional information about the vectors discussed herein.
  • Figure 44 shows the structure of CMVR gag new 5- 1 h (SEQ ID NO: 75).
  • Figure 45 shows the structure of CMVR gag new 5-h (SEQ ID NO: 77).
  • Figure 46 shows the structure of CMVR gag new 6-h (SEQ ID NO: 79). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
  • HIV-I Gag has been included in nearly all HIV vaccines entering clinical trials because of its importance in SIV models and its correlation with protection in HIV- infected long-term non-progressors. However, HIV Gag has proven less immunogenic then Env in phase I clinical trials.
  • a vaccine that includes highly immunogenic Gag will be an important component of an effective HIV vaccine.
  • regions in HIV-I Gag have been identified as contributing to the decreased immunogenicity observed for HIV-I Gag. Replacement of these regions with corresponding SIV sequences significantly increased the resulting T-cell response to HIV Gag in mice. These chimeras may be utilized in an HIV vaccine to significantly enhance the overall immunogenicity of the vaccine.
  • Nucleic acids encoding HIV-I Gag polypeptides comprising a modified amino acid sequence in which one or more amino acids in said HIV-I Gag polypeptide have been replaced with one or more amino acids from the corresponding region in the SIV Gag protein and the corresponding modified HIV-I Gag polypeptides are disclosed herein.
  • the encoded modified HIV-I Gag polypeptides may have a length at least about 95%, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%.
  • the encoded modified HIV-I Gag polypeptide is a fragment of the HIV-I Gag protein which is at least about 10, at least about 20, at least about 30, at least about 40, at least about 60, at least about 80, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 460, at least about 470, at least about 480, at least about 490, or at least about 500 amino acids in length and in which one or more amino acids of the wild type HIV-I sequence has been replaced by one or more amino acids from the corresponding region of the SIV Gag protein.
  • the foregoing fragments of the HIV-I Gag protein comprise one or more antigenic regions capable of inducing an immune response against HIV-I, wherein the at least one of the one or more antigenic regions comprises a modified sequence in which one or more amino acids from the HIV-I Gag polypeptide has been replaced with one or more amino acids from the corresponding region in the SIV Gag protein.
  • the modified HIV-I Gag polypeptides are able to induce a greater immune response than the wild-type HIV-I Gag polypeptide.
  • the immune response against the modified HIV Gag protein may be at least about 1.5 times greater, at least about 5 times greater, or at least about 10 times greater than the immune response against the wild-type HIV-I protein.
  • the modified HIV-I Gag polypeptides induce a greater CD8 T-cell response than the wild-type HIV-I protein.
  • at least about 5 contiguous amino acids, at least about 10 contiguous amino acids, at least about 20 contiguous amino acids, at least about 30 contiguous amino acids, at least about 40 contiguous amino acids, at least about 50 contiguous amino acids, at least about 60 contiguous amino acids or more than 60 contiguous amino acids of the HIV-I Gag polypeptide have been replaced by at least about 5 contiguous amino acids, at least about 10 contiguous amino acids, at least about 20 contiguous amino acids, at least about 30 contiguous amino acids, at least about 40 contiguous amino acids, at least about 50 contiguous amino acids, at least about 60 contiguous amino acids or more than 60 contiguous amino acids of the SIV-I Gag protein.
  • the modified amino acid sequence comprises a sequence in which at least a portion of the carboxy terminal region of the HIV-I Gag polypeptide has been replaced with a portion of the carboxy terminal region from the SIV Gag protein.
  • at least a portion of the 179 carboxy terminal amino acids of the HIV-I Gag polypeptide has been replaced with an amino acid sequence comprising the corresponding portion of the SIV Gag protein.
  • the at least a portion of the 179 carboxy terminal amino acids of the HIV-I Gag polypeptide which has been replaced is at least about 5 contiguous amino acids in length, at least about 10 contiguous amino acids in length, at least about 20 contiguous amino acids in length, at least about 30 contiguous amino acids in length, at least about 40 contiguous amino acids in length, at least about 50 contiguous amino acids in length, at least about 60 contiguous amino acids in length or more than 60 contiguous amino acids in length.
  • the modified HIV-I Gag polypeptide may comprise a modified amino acid sequence in which the carboxy terminal 179 amino acids of the HIV-I Gag polypeptide have been replaced with amino acids 333-510 from the SIV Gag protein.
  • the modified HIV-I Gag polypeptide may comprise a sequence in which at least a portion of the amino acid sequence between amino acids 358-41 1 of the HIV-I Gag polypeptide has been replaced with at least a portion of the corresponding region of the SIV Gag protein.
  • the modified HIV-I Gag polypeptide may comprise a sequence in which amino acids 358-41 1 of the HIV-I Gag polypeptide have been replaced with amino acids 359-412 of the SIV Gag protein.
  • the modified HIV-I Gag polypeptide may comprise a modified amino acid sequence comprising a sequence in which at least a portion of the amino acid sequence between amino acids 419-454 of the HIV-I Gag polypeptide has been replaced with at least a portion of the corresponding region of the SIV Gag protein.
  • the modified HIV- 1 Gag polypeptide may comprise a modified amino acid sequence comprising a sequence in which amino acids 419-454 of the HIV-I Gag polypeptide have been replaced with amino acids 420-457 of the SIV Gag protein.
  • the modified HIV-I Gag polypeptide may comprise a modified amino acid sequence in which at least a portion of the CypA binding domain of HIV-I has been replaced with at least a portion of the SIV-I CypA binding domain.
  • the modified HIV-I Gag polypeptide may comprise a modified amino acid sequence comprising a sequence in which at least a portion of the amino acid sequence between amino acids 214-225 of the HIV-I Gag polypeptide has been replaced with at least a portion of the corresponding region of the SIV Gag protein.
  • the modified HIV-I Gag polypeptide comprises a modified amino acid sequence in which amino acids 214-225 of the HIV-I Gag polypeptide have been replaced with amino acids 216-225 of the SIV Gag protein and amino acids 332-500 of the HIV-I Gag polypeptide have been replaced with amino acids 333-510 of the SIV Gag protein.
  • the modified HIV-I Gag polypeptide may comprise a modified amino acid sequence comprising a sequence in which amino acids 214-254 of the HIV-I Gag polypeptide have been replaced with amino acids 216-255 of the SIV Gag protein and amino acids 332-510 of the HIV-I Gag polypeptide have been replaced with amino acids 333-510 of the SIV Gag protein.
  • the modified HIV-I Gag polypeptide comprises a modified amino acid sequence in which at least a portion of the amino acid sequence between amino acids 358-418 of the HIV-I Gag polypeptide has been replaced with at least a portion of the corresponding region of the SIV Gag protein. In some embodiments, the modified HIV-I Gag polypeptide comprises a modified amino acid sequence in which at least a portion of the amino acid sequence between amino acids 419-462 of the HIV-I Gag polypeptide has been replaced with at least a portion of the corresponding region of the SIV Gag protein .
  • the modified HIV-I Gag polypeptide comprises a plurality of any of the modified amino acid sequences described herein.
  • any of the modified HIV-I Gag polypeptides described herein may be made by means familiar to those skilled in the art.
  • the modified HIV-I proteins may be made by expressing them from nucleic acid vectors.
  • Nucleic acids encoding the modified HIV-I Gag polypeptides may be generated using methodology familiar to those skilled in the art.
  • nucleic acids encoding the modified HIV-I proteins may be made by digesting a nucleic acid encoding the wild-type HIV-I protein with appropriate restriction enzymes and inserting a nucleic acid encoding the desired SIV amino acid sequence at the restriction sites.
  • nucleic acids encoding the modified HIV-I Gag polypeptides may be made using site-directed mutagenesis or PCR-mediated mutagenesis. It will be appreciated that other techniques may also be used to prepare nucleic acids the modified HIV-I Gag polypeptides.
  • Vectors comprising nucleic acids encoding the modified HIV-I polypeptides are also provided.
  • the vector encoding any of the modified HIV-I Gag polypeptides described herein may be a viral vector.
  • the viral vector may be an adenovirus vector.
  • the adenovirus vector may be an adenovirus 5 vector or an adenovirus 26 vector.
  • the vector is a gene-based vector.
  • the vector is a plasmid DNA vector used alone or in combination with an adjuvant.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non- episomal mammalian vectors
  • Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "expression vectors".
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the embodiments described herein are intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • the recombinant expression vectors of the invention comprise a nucleic acid encoding any of the modified HIV-I Gag polypeptides described herein in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
  • operably linked is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990), the disclosure of which is incorporated herein by reference in its entirety.
  • Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce any of the modified HIV-I Gag polypeptides described herein.
  • the recombinant expression vectors described herein can be designed for expression of any of the modified HIV-I Gag polypeptides described herein.
  • any of the modified HIV-I Gag polypeptides described herein can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells, or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990), the disclosure of which is incorporated herein by reference in its entirety.
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31- 40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N. J.), the disclosures of which are incorporated herein by reference in their entireties, which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
  • GST glutathione S-transferase
  • Purified fusion or non-fusion proteins can be utilized in any of the assays described herein for assessing the immunogenicity of any of the modified HIV-I Gag polypeptides described herein or in the procedures for inducing an immune response against HIV-I in an individual described herein.
  • Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET Hd (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89), the disclosures of which are incorporated herein by reference in their entireties.
  • Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter.
  • Target gene expression from the pET 1 Id vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gn 1).
  • This viral polymerase is supplied by host strains BL21 (DE3) or HMS174(DE3) from a resident prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacterium with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128, the disclosure of which is incorporated herein by reference in its entirety).
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., (1992) Nucleic Acids Res. 20:21 11-21 18, the disclosure of which is incorporated herein by reference in its entirety).
  • Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the vector encoding any of the modified HIV-I Gag polypeptides described herein is a yeast expression vector.
  • yeast expression vectors for expression in yeast S. cerivisae include pYepSec 1 (Baldari, et al., (1987) Embo J. 6:229- 234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:1 13-123), pYES2 (Invitrogen Corporation, San Diego, Calif), and picZ (InVitrogen Corp, San Diego, Calif), the disclosures of which are incorporated herein by reference in their entireties.
  • any of the modified HIV-I Gag polypeptides described herein can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al. (1983) MoI. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39), the disclosures of which are incorporated herein by reference in their entireties.
  • the modified HIV-I Gag polypeptides are expressed according to Karniski et al, Am. J. Physiol. (1998) 275: F79- 87, the disclosure of which is incorporated herein by reference in its entirety.
  • any of the modified HIV-I Gag polypeptides described herein may be expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195), the disclosures of which are incorporated herein by reference in their entireties.
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art, and are further described below.
  • any of the modified HIV-I Gag polypeptides described herein may be expressed from vectors comprising a CMV enhancer, an HTLV-I R region and a CMV IE splicing acceptor operably linked to a nucleic acid encoding the modified HIV-I Gag polypeptide.
  • any of the modified HIV-I Gag polypeptides described herein may be expressed from the vectors described in U.S. Patent No. 7,094,598, the disclosure of which is incorporated herein by reference in its entirety.
  • polypeptides lacking one or more of these SIV epitopes or nucleic acids encoding such polypeptides may be used in any of the compositions and methods described herein
  • Exemplary nucleic acids encoding polypeptides lacking these epitopes are provided as SEQ ID NOs.: 75, 77, and 79.
  • Exemplary polypeptides lacking these epitopes are provided as SEQ ID NOs. 76, 78 and 80.
  • Another aspect of the invention pertains to host cells into which a nucleic acid, such as any of the vectors described herein, encoding any of the modified HIV-I Gag polypeptides described herein has been introduced.
  • host cell and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • any of the modified HIV-I Gag polypeptides described herein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells or human cells).
  • bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells or human cells).
  • CHO Chinese hamster ovary cells
  • COS cells or human cells include mouse 3T3 cells as further described in the Examples.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989, the disclosure of which is incorporated herein by reference in its entirety), and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a THAP-family protein or can be introduced on a separate vector.
  • a host cell such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) any of the modified HIV-I Gag polypeptides described herein Accordingly, the invention further provides methods for producing a modified HIV-I Gag polypeptide using the host cells of the invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a modified HIV-I Gag polypeptide has been introduced) in a suitable medium such that a modified HIV-I Gag polypeptide is produced.
  • the method further comprises isolating a modified HIV-I Gag polypeptide from the medium or the host cell.
  • Another embodiment provides methods comprising providing a cell capable of expressing any of the modified HIV-I Gag polypeptides described herein, culturing said cell in a suitable medium such that a modified HIV-I Gag polypeptide is produced, and isolating or purifying the modified HIV-I Gag polypeptide from the medium or cell.
  • the nucleic acid vectors for producing or administering any of the modified HIV-I Gag polypeptides described herein comprise regulatory elements (e.g. promotor, enhancer, etc) capable of directing the expression of the modified HIV-I Gag polypeptide in the targeted cell.
  • regulatory elements e.g. promotor, enhancer, etc
  • the human cytomegalovirus (CMV) immediate early gene promoter can be used to obtain high-level expression of any of the modified HIV-I Gag polypeptides described herein.
  • CMV cytomegalovirus
  • the use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose.
  • a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized.
  • Selection of a promoter that is regulated in response to specific physiologic or synthetic signals can permit inducible expression of the gene product.
  • expression of a transgene, or transgenes when a multicistronic vector is utilized is toxic to the cells in which the vector is produced in, it may be desirable to prohibit or reduce expression of one or more of the transgenes.
  • inducible promoter systems are available for production of viral vectors where the transgene product may be toxic.
  • the ecdysone system (Invitrogen, Carlsbad, CA) is one such system. This system is designed to allow regulated expression of a gene of interest in mammalian cells. It consists of a tightly regulated expression mechanism that allows virtually no basal level expression of the transgene, but over 200-fold inducibility.
  • the system is based on the heterodimeric ecdysone receptor of Drosophila, and when ecdysone or an analog such as muristerone A binds to the receptor, the receptor activates a promoter to turn on expression of the downstream transgene high levels of mRNA transcripts are attained.
  • both monomers of the heterodimeric receptor are constitutively expressed from one vector, whereas the ecdysone-responsive promoter which drives expression of the gene of interest is on another plasmid.
  • Engineering of this type of system into the gene transfer vector of interest would therefore be useful.
  • Cotransfection of plasmids containing the gene of interest and the receptor monomers in the producer cell line would then allow for the production of the gene transfer vector without expression of a potentially toxic transgene.
  • expression of the transgene could be activated with ecdysone or muristeron A.
  • Tet-Off or Tet On system (Clontech, Palo Alto, CA) originally developed by Gossen and Bujard (Gossen and Bujard, 1992; Gossen et al, 1995).
  • This system also allows high levels of gene expression to be regulated in response to tetracycline or tetracycline derivatives such as doxycycline.
  • Tet-On system gene expression is turned on in the presence of doxycycline
  • Tet-Off system gene expression is turned on in the absence of doxycycline.
  • the tetracycline operator sequence to which the tetracycline repressor binds, and the tetracycline repressor protein is cloned into a plasmid behind a promoter that has tetracycline-responsive elements present in it.
  • a second plasmid contains a regulatory element called the tetracycline-controlled transactivator, which is composed, in the Tet Off system, of the VP 16 domain from the herpes simplex virus and the wild-type tertracycline repressor.
  • a transgene in a gene therapy vector it may be desirable to regulate expression of a transgene in a gene therapy vector.
  • different viral promoters with varying strengths of activity may be utilized depending on the level of expression desired.
  • the CMV immediate early promoter is often used to provide strong transcriptional activation. Modified versions of the CMV promoter that are less potent have also been used when reduced levels of expression of the transgene are desired.
  • retroviral promoters such as the LTRs from MLV or MMTV are often used.
  • viral promoters that may be used depending on the desired effect include SV40, RSV LTR, HIV-I and HfV-2 LTR, adenovirus promoters such as from the EIA, E2A, or MLP region, AAV LTR, cauliflower mosaic virus, HSV-TK, and avian sarcoma virus.
  • tissue specific promoters may be used to effect transcription in specific tissues or cells so as to reduce potential toxicity or undesirable effects to non- targeted tissues.
  • promoters such as the PSA, probasin, prostatic acid phosphatase or prostate-specific glandular kallikrein (hK2) may be used to target gene expression in the prostate.
  • promoters as follows may be used to target gene expression in other tissues.
  • Tissue specific promoters include in (a) pancreas: insulin, elastin, amylase, pdr-I, pdx-I, glucokinase; (b) liver: albumin PEPCK, HBV enhancer, alpha fetoprotein, apolipoprotein C, alpha-I antitrypsin, vitellogenin, NF-AB, Transthyretin; (c) skeletal muscle: myosin H chain, muscle creatine kinase, dystrophin, calpain p94, skeletal alpha-actin, fast troponin 1; (d) skin: keratin K6, keratin KI; (e) lung: CFTR, human cytokeratin IS (K 18), pulmonary surfactant proteins A, B and C, CC-IO, Pi; (f) smooth muscle: sm22 alpha, SM-alpha-actin; (g) endothelium: endothelin- I
  • promoters as those that are hormone or cytokine regulatable.
  • promoters that are hormone regulatable include MMTV, MT-I, ecdysone and RuBisco.
  • Other hormone regulated promoters such as those responsive to thyroid, pituitary and adrenal hormones are expected to be useful in the present invention.
  • Cytokine and inflammatory protein responsive promoters that could be used include K and T Kininogen (Kageyama et al., 1987), c-fos, TNF-alpha, C-reactive protein (Arcone et al., 1988), haptoglobin (Oliviero et al., 1987), serum amyloid A2, C/EBP alpha, IL-I, IL-6 (PoIi and Cortese, 1989), Complement C3 (Wilson et al., 1990), IL-8, alpha- 1 acid glycoprotein (Prowse and Baumann, 1988), alpha- 1 antitypsin, lipoprotein lipase (Zechner et al., 1988), angiotensinogen (Ron et al., 1991), fibrinogen, c-jun (inducible by phorbol esters, TNF alpha, UV radiation, retinoic acid, and hydrogen peroxide), collagenase (induced by phorbol esters and reti
  • cell cycle regulatable promoters may be useful in the present invention.
  • a strong CMV promoter to drive expression of a first gene such as pi 6 that arrests cells in the Gl phase could be followed by expression of a second gene such as p53 under the control of a promoter that is active in the Gl phase of the cell cycle, thus providing a "second hit" that would push the cell into apoptosis.
  • Other promoters such as those of various cyclins, PCNA, galectin-3, E2FI, p53 and BRCAI could be used.
  • the nucleic acids for producing or administering any of the modified HIV-I Gag polypeptides described herein may contain one or more enhancers.
  • Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements.
  • a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.
  • promoters additional to the tissue specific promoters listed above, cellular promoters/enhancers and inducible promoters/enhancers that could be used in combination with the nucleic acid encoding a gene of interest in an expression construct (list of enhancers, and Table 1). Additionally, any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of the gene. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.
  • Suitable enhancers include: Immunoglobulin Heavy Chain; Immunoglobulin Light Chain; T-CeIl Receptor; HLA DQ (x and DQ beta; beta-Interferon; Interleukin-2; Interleukin-2 Receptor; MHC Class II 5; MHC Class II HLA-DRalpha; beta- Actin; Muscle Creatine Kinase; Prealbumin (Transthyretin); Elastase I; Metallothionein; Collagenase; Albumin Gene; alpha-Fetoprotein; -Globin; beta-Globin; e-fos; c-HA-ras; Insulin; Neural Cell Adhesion Molecule (NCAM); alpha a 1 -Antitrypsin; H2B (TH2B) Histone; Mouse or Type I Collagen; Glucose-Regulated Proteins (GRP94 and GRP78); Rat Growth Hormone; Human Serum Amyloid A (SAA); Tropot al
  • any of the modified HIV-I Gag polypeptides described herein may be produced or administered via a vector comprising a virus or engineered construct derived from a viral genome.
  • a vector comprising a virus or engineered construct derived from a viral genome.
  • the first viruses used as gene vectors were DNA viruses including the papovaviruses (simian virus 40, bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). These have a relatively low capacity for foreign DNA sequences and have a restricted host spectrum.
  • papovaviruses simian virus 40, bovine papilloma virus, and polyoma
  • adenoviruses Rosgeway, 1988; Baichwal and Sugden, 1986.
  • the modified HIV-I Gag polypeptides are produced or administered using a viral vector.
  • the viral vector may be an adenovirus vector.
  • the adenovirus vector may be an adenovirus 5 vector.
  • the adenovirus vector may be an adenovirus 26 vectors.
  • Nucleic acids encoding any of the modified HIV-I Gag polypeptides described herein may be operably linked to a polyadenylation signal to effect proper polyadenylation of the gene transcript.
  • the nature of the polyadenylation signal is not believed to be crucial and any such sequence may be employed such as human or bovine growth hormone and SV40 polyadenylation signals.
  • vectors for expressing any of the modified HIV-I Gag polypeptides described herein may include a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
  • a nucleic acid encoding any of the modified HIV-I Gag polypeptides described herein is incorporated into a viral infectious particle to mediate gene transfer to a cell.
  • retroviral or bovine papilloma virus may be employed, both of which permit permanent transformation of a host cell with a gene(s) of interest.
  • viral infection of cells is used in order to deliver therapeutically significant genes to a cell.
  • the virus simply will be exposed to the appropriate host cell under physiologic conditions, permitting uptake of the virus.
  • adenovirus is exemplified, the present methods may be advantageously employed with other viral or non-viral vectors, as discussed below.
  • Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized DNA genome, ease of manipulation, high titer, wide target-cell range, and high infectivity.
  • the roughly 36 kB viral genome is bounded by 100-200 base pair (bp) inverted terminal repeats (ITR), in which are contained cis acting elements necessary for viral DNA replication and packaging.
  • ITR inverted terminal repeats
  • the early (E) and late (L) regions of the genome that contain different transcription units are divided by the onset of viral DNA replication.
  • the El region (EIA and EIB) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes.
  • the expression of the E2 region results in the synthesis of the proteins for viral DNA replication.
  • MLP major late promoter
  • adenovirus In order for adenovirus to be optimized for gene therapy, it is necessary to maximize the carrying capacity so that large segments of DNA can be included. It also is very desirable to reduce the toxicity and immunologic reaction associated with certain adenoviral products. The two goals are, to an extent, coterminous in that elimination of adenoviral genes serves both ends. By practice of the present invention, it is possible achieve both these goals while retaining the ability to manipulate the therapeutic constructs with relative case. [0117] The large displacement of DNA is possible because the cis elements required for viral DNA replication all are localized in the inverted terminal repeats (ITR) (100-200 bp) at either end of the linear viral genome. Plasmids containing ITR's can replicate in the presence of a non-defective adenovirus (Hay et al., 1984). Therefore, inclusion of these elements in an adenoviral vector should permit replication.
  • ITR inverted terminal repeats
  • the packaging signal for viral encapsidation is localized between 194 385 bp (0.5-1.1 map units) at the left end of the viral genome (Hearing et al., 1987).
  • This signal mimics the protein recognition site in bacteriophage k DNA where a specific sequence close to the left end, but outside the cohesive end sequence, mediates the binding to proteins that are required for insertion of the DNA into the head structure.
  • El substitution vectors of Ad have demonstrated that a 450 bp (0-1.25 map units) fragment at the left end of the viral genome could direct packaging in 293 cells (Levrero et al., 1991).
  • adenoviral genome can be incorporated into the genome of mammalian cells and the genes encoded thereby expressed. These cell lines are capable of supporting the replication of an adenoviral vector that is deficient in the adenoviral function encoded by the cell line. There also have been reports of complementation of replication deficient adenoviral vectors by "helping" vectors, e.g., wild-type virus or conditionally defective mutants.
  • Replication-deficient adenoviral vectors can be complemented, in trans, by helper virus. This observation alone does not permit isolation of the replication-deficient vectors, however, since the presence of helper virus, needed to provide replicative functions, would contaminate any preparation. Thus, an additional element was needed that would add specificity to the replication and/or packaging of the replication-deficient vector. That element, as provided for in the present invention, derives from the packaging function of adenovirus.
  • helper viruses that are packaged with varying efficiencies.
  • the mutations are point mutations or deletions.
  • helper viruses with low efficiency packaging are grown in helper cells, the virus is packaged, albeit at reduced rates compared to wild-type virus, thereby permitting propagation of the helper.
  • helper viruses are grown in cells along with virus that contains wild-type packaging signals, however, the wild-type packaging signals are recognized preferentially over the mutated versions.
  • the virus containing the wild-type signals are packaged selectively when compared to the helpers. If the preference is great enough, stocks approaching homogeneity should be achieved.
  • the adenovirus vectors encoding any of the modified HIV-I Gag polypeptides described herein may be adenovirus 5 or adenovirus 26 vectors.
  • retrovirus vectors may be used to produce or administer any of the modified HIV-I Gag polypeptides described herein.
  • the retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990). The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins.
  • the integration results in the retention of the viral gene sequences in the recipient cell and its descendants.
  • the retroviral genome contains three genes - gag, pol and env - that code for capsid proteins, polymerase enzyme, and envelope components, respectively.
  • Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and also are required for integration in the host cell genome (Coffin, 1990).
  • a nucleic acid encoding a promoter is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective.
  • a packaging cell line containing the gag, pol and env genes but without the LTR and T components is constructed (Mann et al., 1983).
  • Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression of many types of retroviruses require the division of host cells (Paskind et al., 1975).
  • adenoassociated virus (AAV) vectors may be used to produce or administer any of the modified HIV-I Gag polypeptides described herein.
  • AAV utilizes a linear, single-stranded DNA of about 4700 base pairs. Inverted terminal repeats flank the genome. Two genes are present within the genome, giving rise to a number of distinct gene products. The first, the cap gene, produces three different virion proteins (VP), designated VP-I, VP 2 and VP-3. [0130] The second, the rep gene, encodes four non-structural proteins (NS). One or more of these rep gene products is responsible for transactivating AAV transcription.
  • VP virion proteins
  • NS non-structural proteins
  • the three promoters in AAV are designated by their location, in map units, in the genome. These are, from left to right, p5, pl9 and p40. Transcription gives rise to six transcripts, two initiated at each of three promoters, with one of each pair being spliced.
  • the splice site derived from map units 42-46, is the same for each transcript.
  • the four non-structural proteins apparently are derived from the longer of the transcripts, and three virion proteins all arise from the smallest transcript.
  • AAV is not associated with any pathologic state in humans. Interestingly, for efficient replication, AAV requires "helping" functions from viruses such as herpes simplex virus I and II, cytomegalovirus, pseudorabies virus and, of course, adenovirus.
  • helpers The best characterized of the helpers is adenovirus, and many "early" functions for this virus have been shown to assist with AAV replication. Low level expression of AAV rep proteins is believed to hold AAV structural expression in check, and helper virus infection is thought to remove this block.
  • the terminal repeats of the AAV vector can be obtained by restriction endonuclease digestion of AAV or a plasmid such as p201, which contains a modified AAV genome (Samulski et al, 1987), or by other methods known to the skilled artisan, including but not limited to chemical or enzymatic synthesis of the terminal repeats based upon the published sequence of AAV.
  • the ordinarily skilled artisan can determine, by well-known methods such as deletion analysis, the minimum sequence or part of the AAV ITRs which is required to allow function, i.e., stable and site specific integration.
  • AAV-based vectors have proven to be safe and effective vehicles for gene delivery in vitro, and these vectors are being developed and tested in pre-clinical and clinical stages for a wide range of applications in potential gene therapy, both ex vivo and in vivo (Carter and Flotte, 1996; Chattedee et al., 1995; Ferrari et al., 1996; Fisher et al., 1996; Flotte et al., 1993; Goodman et al., 1994; Kaplitt et al., 1994; 1996, Kessler et al., 1996; Koeberl et al., 1997; Mizukami et al., 1996; Xiao et al., 1996, the disclosures of which are incorporated herein by reference in their entireties).
  • AAV-mediated efficient gene transfer and expression in the lung has led to clinical trials for the treatment of cystic fibrosis (Carter and Flotte, 1996; Flotte et al., 1993, the disclosures of which are incorporated herein by reference).
  • the prospects for treatment of muscular dystrophy by AAV-mediated gene delivery of the dystrophin gene to skeletal muscle, of Parkinson's disease by tyrosine hydroxylase gene delivery to the brain, of hemophilia B by Factor IX gene delivery to the liver, and potentially of myocardial infarction by vascular endothelial growth factor gene to the heart appear promising since AAV- mediated transgene expression in these organs has recently been shown to be highly efficient (Fisher et al., 1996; Flotte et al., 1993; Kaplitt et al., 1994; 1996; Koeberl et al., 1997; McCown et al., 1996; Ping et al., 1996; and Xiao et
  • viral vectors may be employed to produce or administer any of the modified HIV-I proteins described herein.
  • Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988) and hepatitus B viruses have also been developed and are useful in the present invention. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; and Horwich et al., 1990, the disclosures of which are incorporated herein by reference in their entireties.).
  • the nucleic acids encoding any of the modified HIV-I Gag polypeptides described herein are housed within an infective virus that has been engineered to express a specific binding ligand.
  • the virus particle will thus bind specifically to the cognate receptors of the target cell and deliver the contents to the cell.
  • a novel approach designed to allow specific targeting of retrovirus vectors was recently developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification can permit the specific infection of hepatocytes via sialoglycoprotein receptors.
  • non-viral methods are used to produce or administer nucleic acids encoding any of the modified HIV-I Gag polypeptides described herein.
  • DNA constructs of the present invention are generally delivered to a cell.
  • the nucleic acid to be transferred is non-infectious, and can be transferred using non-viral methods.
  • the nucleic acid encoding the modified HIV-I Gag polypeptide may be positioned and expressed at different sites.
  • the nucleic acid encoding the therapeutic gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation).
  • the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle.
  • the expression construct may be entrapped in a liposome.
  • Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991).
  • DNA-lipid complexes are potential non-viral vectors for use in gene therapy.
  • Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful.
  • Wong et al. (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa, and hepatoma cells.
  • Nicolau et al. (1987) accomplished successful liposome-mediated gene transfer in rats after intravenous injection. Also included are various commercial approaches involving "lipofection" technology.
  • the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al., 1989).
  • HVJ hemagglutinating virus
  • the liposome may be complexed or employed in conjunction with nuclear nonhistone chromosomal proteins (HMG-I) (Kato et al., 1991).
  • HMG-I nuclear nonhistone chromosomal proteins
  • the liposome may be complexed or employed in conjunction with both HVJ and HMG-I. In that such expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present invention.
  • receptor-mediated delivery vehicles which can be employed to deliver a nucleic acid encoding any of the modified HIV-I Gag polypeptides described herein into cells. These take advantage of the selective uptake of macromolecules by receptor mediated endocytosis in almost all eukaryotic cells. Because of the cell type specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993).
  • Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent.
  • ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and transferring (Wagner et al., 1990).
  • the delivery vehicle may comprise a ligand and a liposome.
  • a ligand and a liposome For example, Nicolau et al, (1987) employed lactosyl-ceramide, a galactose terminal asialganglioside, incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes.
  • a nucleic acid encoding a therapeutic gene also may be specifically delivered into a cell type such as prostate, epithelial or tumor cells, by any number of receptor-ligand systems with or without liposomes.
  • the human prostate-specific antigen (Watt et al, 1986) may be used as the receptor for mediated delivery of a nucleic acid in prostate tissue.
  • the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is applicable particularly for transfer in vitro, however, it may be applied for in vivo use as well. Dubensky et al, (1984) successfully injected polyornavirus DNA in the form of CaP04 precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection.
  • Another embodiment for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al, 1987). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical cur-rent, which in turn provides the motive force (Yang et al, 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.
  • Some embodiments relate to methods for inducing an immune response to HIV-I comprising administering any of the modified HIV-I Gag polypeptides described herein to an individual.
  • the modified HIV-I Gag polypeptides are administered via a nucleic acid encoding the modified HIV-I Gag polypeptide.
  • the nucleic acids may be any of those described herein or other suitable nucleic acids.
  • the modified HIV-I Gag polypeptides are administered in addition to other proteins which induce an immune response against HIV-I .
  • the other proteins may be other HIV-I proteins.
  • the other proteins may be Pol, Nef and Env.
  • the modified HIV-I Gag polypeptides and one or more of the other proteins which induce an immune response against HIV-I may be administered as fusion proteins.
  • the modified HIV-I Gag polypeptides and one or more of the other proteins which induce an immune response against HIV-I may be administered as separate proteins.
  • compositions capable of eliciting an immune response against HIV can be capable of eliciting a protective immune response against HIV when administered alone or in combination with at least one additional immunogenic compositions.
  • the ability to produce an immune response after exposure to an antigen is a function of complex cellular and humoral processes, and that different subjects have varying capacity to respond to an immunological stimulus.
  • compositions disclosed herein are capable of eliciting an immune response in an immunocompetent subject, that is a subject that is physiologically capable of responding to an immunological stimulus by the production of a substantially normal immune response, e.g., including the production of antibodies that specifically interact with the immunological stimulus, and/or the production of functional T cells (CD4 + and/or CD8 + T cells) that bear receptors that specifically interact with the immunological stimulus.
  • a substantially normal immune response e.g., including the production of antibodies that specifically interact with the immunological stimulus, and/or the production of functional T cells (CD4 + and/or CD8 + T cells) that bear receptors that specifically interact with the immunological stimulus.
  • compositions to a subject prior to exposure to HIV (that is, prophylactically, e.g., as a vaccine) or therapeutically at a time following exposure to HIV during which the subject is nonetheless capable of developing an immune response to a stimulus, such as an antigenic polypeptide.
  • compositions may comprise a nucleic acid encoding any of the modified Gag HIV-I polypeptides described herein or the compositions may comprise the modified HIV-I Gag polypeptide itself.
  • the modified Gag HIV-I polypeptides When administered in conjunction with nucleic acids encoding one or more other polypeptides capable of inducing an immune response against HIV-I, the modified Gag HIV-I polypeptides may be encoded by the same nucleic acid as the one or more other polypeptides. Alternatively, the modified HIV-I polypeptide may be encoded on a separate nucleic acid from the one or more other polypeptides.
  • the modified HIV-I Gag polypeptide itself is administered with one or more other polypeptides which induce an immune response against HIV-I
  • the modified HIV-I Gag polypeptide may be fused to one or more of the other polypeptides.
  • the modified HIV-I Gag polypeptide may be administered as a separate polypeptide from the one or more other polypeptides.
  • the compositions when formulated for administration to a subject, also include a pharmaceutically acceptable carrier or excipient, for example, an aqueous carrier, such as phosphate buffered saline (PBS) or another neutral physiological salt solution.
  • a pharmaceutically acceptable carrier or excipient for example, an aqueous carrier, such as phosphate buffered saline (PBS) or another neutral physiological salt solution.
  • PBS phosphate buffered saline
  • the composition can also include an adjuvant or other immunostimulatory molecule.
  • the composition can be administered one or more times to a subject to elicit an immune response.
  • the composition can be administered multiple times at intervals of at least about 28 days, or at different intervals as dictated by logistical or therapeutic concerns.
  • compositions or medicaments for the therapeutic or prophylactic treatment of an HIV infection includes pharmaceutical compositions or medicaments for the therapeutic or prophylactic treatment of an HIV infection.
  • the use of the compositions disclosed herein in the production of medicament for the therapeutic or prophylactic treatment of HIV is also expressly contemplated. Any of the limitations or formulations disclosed above with respect to compositions are applicable to their use in or as medicaments for the treatment of an HIV infection.
  • compositions described above relate to methods for eliciting an immune response against HIV by administering the compositions described above to an individual.
  • the individual is a human subject.
  • the composition may comprise any of the nucleic acids described herein encoding any of the modified HIV-I Gag polypeptides described herein.
  • the composition may comprise any of the modified HIV-I Gag polypeptides described herein.
  • compositions can be administered to a subject to elicit an immune response with desired characteristics, including the production of HIV specific antibodies, or the production of functional T cells that react with HIV.
  • the T cells may be CD8 T cells.
  • the compositions may be administered using any suitable route.
  • the composition is administered intramuscularly, for example, using a syringe or needleless delivery device.
  • the composition is administered by other routes, such as intravenous, transdermal, intranasal, oral (or via another mucosa).
  • the compositions comprise any of the viral vectors described herein.
  • the viral vectors are adenoviral vectors (for example a replication deficient adenoviral vectors).
  • the adenovirus vector is an adenovirus 5 vector.
  • the viral vector is an adenovirus 26 vector.
  • one or more doses of a "primer” composition can be administered to a subject, followed by administration of one or more doses of a "booster” composition including viral particles comprising any of the viral vectors described above.
  • the "primer” composition can comprise any of the vectors described above as naked DNA.
  • the "booster” composition can comprise adenoviral particles comprising any of the adenovirus vectors described above.
  • compositions for inducing an immune response comprise a pharmaceutically acceptable carrier or excipient.
  • pharmaceutically acceptable carriers or excipients of use are conventional. Remington 's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the polypeptides and polynucleotides disclosed herein.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like
  • solid compositions for example, powder, pill, tablet, or capsule forms
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • the modified HIV-I Gag polypeptides may be used, for example, as pharmaceutical compositions (medicaments) for use in therapeutic, for example, prophylactic regimens (e.g., vaccines) and administered to subjects (e.g., human subjects) to elicit an immune response against HIV-I.
  • the compositions described herein can be administered to a human (or non-human) subject prior to infection with HIV to inhibit infection by or replication of the virus.
  • the pharmaceutical compositions described above can be administered to a subject to elicit a protective immune response against HIV.
  • a therapeutically effective (e.g., immunologically effective) amount of the nucleic acid constructs are administered to a subject, such as a human (or non-human) subject.
  • a "therapeutically effective amount” is a quantity of a chemical composition (such as a nucleic acid construct, vector, or polypeptide) used to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to express an adequate amount of antigen to elicit an antibody or T cell response, or to inhibit or prevent infection by or replication of the virus, or to prevent, lessen or ameliorate symptoms caused by infection with the virus.
  • a dosage When administered to a subject, a dosage will generally be used that will achieve target tissue or systemic concentrations that are empirically determined to achieve an in vitro effect. Such dosages can be determined without undue experimentation by those of ordinary skill in the art. Exemplary dosages are described in detail in the Examples.
  • a pharmaceutical composition including a nucleic acid encoding any of the modified HIV-I Gag polypetides described herein or the modified HIV-I Gag polypeptide itself can be administered by any means known to one of skill in the art (see Banga, A., "Parenteral Controlled Delivery of Therapeutic Peptides and Proteins," in Therapeutic Peptides and Proteins, Technomic Publishing Co., Inc., Lancaster, PA, 1995; DNA Vaccines: Methods and Protocols (Methods in Molecular Medicine) by Douglas B. Lowrie and Robert G. Whalen (Eds.), Humana Press, 2000) such as by intramuscular, subcutaneous, or intravenous injection, but even oral, nasal, or anal administration is contemplated.
  • administration is by subcutaneous or intramuscular injection.
  • Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remingtons Phamaceutical Sciences, 19 th Ed., Mack Publishing Company, Easton, Pennsylvania, 1995.
  • Suitable formulations for the compositions comprising any of the modified HIV-I Gag polypeptides described herein or a nucleic acid encoding any of the modified HIV-I Gag polypeptides described herein, for example, the primer or booster compositions disclosed herein include aqueous and non-aqueous solutions, isotonic sterile solutions, which can contain anti-oxidants, buffers, and bacteriostats, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, immediately prior to use.
  • sterile liquid carrier for example, water
  • Extemporaneous solutions and suspensions can be prepared from sterile powders, granules, and tablets.
  • the carrier is a buffered saline solution.
  • the composition for use in the inventive method is formulated to protect the nucleic acid constructs from damage prior to administration.
  • the composition can be formulated to reduce loss of the adenoviral vectors on devices used to prepare, store, or administer the expression vector, such as glassware, syringes, or needles.
  • the compositions can be formulated to decrease the light sensitivity and/or temperature sensitivity of the components.
  • the composition preferably comprises a pharmaceutically acceptable liquid carrier, such as, for example, those described above, and a stabilizing agent selected from the group consisting of polysorbate 80, L-arginine, polyvinylpyrrolidone, trehalose, and combinations thereof.
  • a pharmaceutically acceptable liquid carrier such as, for example, those described above
  • a stabilizing agent selected from the group consisting of polysorbate 80, L-arginine, polyvinylpyrrolidone, trehalose, and combinations thereof.
  • adenoviral vector-containing compositions are further described in, for example, U.S. Patent 6,225,289, 6,514,943, U.S. Patent Application Publication No. 2003/0153065 Al, and International Patent Application Publication WO 00/34444, the disclosures of which are incorporated herein by reference in their entireties.
  • An adenoviral vector composition also can be formulated to enhance transduction efficiency.
  • the composition can comprise other therapeutic or biologically-active agents.
  • factors that control inflammation such as ibuprofen or steroids, can be part of the adenoviral vector composition to reduce swelling and inflammation associated with in vivo administration of the adenoviral vectors.
  • immune system stimulators can be administered to enhance any immune response to the antigens.
  • Antibiotics i.e., microbicides and fungicides, can be present to treat existing infection and/or reduce the risk of future infection, such as infection associated with gene transfer procedures.
  • compositions can be administered for therapeutic treatments.
  • a therapeutically effective amount of the composition is administered to a subject prior to or following exposure to or infection by HIV.
  • the therapeutic application can be referred to as a prophylactic administration (e.g., a vaccine).
  • a prophylactic administration e.g., a vaccine
  • Single or multiple administrations of the compositions are administered depending on the dosage and frequency as required and tolerated by the subject.
  • the dosage is administered once as a bolus, but in another embodiment can be applied periodically until a therapeutic result, such as a protective immune response, is achieved.
  • the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the subject.
  • Systemic or local administration can be utilized.
  • Controlled release parenteral formulations can be made as implants, oily injections, or as particulate systems.
  • Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Particles, microspheres, and microcapsules smaller than about 1 ⁇ m are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively.
  • Capillaries have a diameter of approximately 5 ⁇ m so that only nanoparticles are administered intravenously. Microparticles are typically around 100 ⁇ m in diameter and are administered subcutaneously or intramuscularly (see, Kreuter, Colloidal Drug Delivery Systems, J.
  • the pharmaceutical composition includes an adjuvant.
  • An adjuvant can be a suspension of minerals, such as alum, aluminum hydroxide, aluminum phosphate, on which antigen is adsorbed; or water-in-oil emulsion in which antigen solution is emulsified in oil (MF-59, Freund's incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibits degradation of antigen and/or causes influx of macrophages).
  • nucleic acid vaccines naturally occurring or synthetic immunostimulatory compositions that bind to and stimulate receptors involved in innate immunity can be administered along with nucleic acid constructs encoding any of the modified HIV-I Gag polypeptides described herein.
  • agents that stimulate certain Toll-like receptors can be administered in combination with the nucleic acid constructs encoding HIV antigenic polypeptides.
  • the nucleic acid construct is administered in combination with immunostimulatory CpG oligonucleotides.
  • nucleic acid constructs encoding any of the modified HIV-I Gag polypeptides described herein can be introduced in vivo as naked DNA plasmids.
  • DNA vectors can be introduced into the desired host cells by methods known in the art, including but not limited to transfection, electroporation (e.g., transcutaneous electroporation), microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (See e.g., Wu et al. J. Biol. Chem., 267:963-967, 1992; Wu and Wu J. Biol.
  • a needleless delivery device such as a BIOJECTOR® needleless injection device can be utilized to introduce the therapeutic nucleic acid constructs in vivo.
  • Receptor- mediated DNA delivery approaches can also be used (Curiel et al. Hum. Gene Ther., 3: 147- 154, 1992; and Wu and Wu, J. Biol. Chem., 262:4429-4432, 1987).
  • a nucleic acid in vivo, is also useful for facilitating transfection of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., WO95/21931), peptides derived from DNA binding proteins (e.g., WO96/25508), or a cationic polymer (e.g., WO95/21931), the disclosures of which are incorporated herein by reference in their entireties.
  • a cationic oligopeptide e.g., WO95/21931
  • peptides derived from DNA binding proteins e.g., WO96/25508
  • a cationic polymer e.g., WO95/21931
  • electroporation can be utilized conveniently to introduce nucleic acid constructs encoding any of the modified HIV-I Gag polypeptides described herein into cells. Electroporation is well known by those of ordinary skill in the art (see, for example: Lohr et al. Cancer Res. 61 :3281-3284, 2001 ; Nakano et al. Hum Gene Ther. 12:1289-1297, 2001; Kim et al. Gene Ther. 10:1216-1224, 2003; Dean et al. Gene Ther. 10:1608-1615, 2003; and Young et al. Gene Ther 10:1465-1470, 2003).
  • a high concentration of vector DNA is added to a suspension of host cell (such as isolated autologous peripheral blood or bone marrow cells) and the mixture shocked with an electrical field.
  • Transcutaneous electroporation can be utilized in animals and humans to introduce heterologous nucleic acids into cells of solid tissues (such as muscle) in vivo.
  • the nucleic acid constructs are introduced into tissues in vivo by introducing a solution containing the DNA into a target tissue, for example, using a needle or trochar in conjunction with electrodes for delivering one or more electrical pulses.
  • a series of electrical pulses can be utilized to optimize transfection, for example, between 3 and ten pulses of 100V and 50 msec. In some cases, multiple sessions or administrations are performed.
  • Biolistic transformation is commonly accomplished in one of several ways.
  • One common method involves propelling inert or biologically active particles at cells. This technique is disclosed in, e.g., U.S. Pat. Nos. 4,945,050, 5,036,006; and 5,100,792, all to Sanford et al., the disclosures of which are hereby incorporated by reference in their entireties.
  • this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof.
  • the plasmid can be introduced into the cell by coating the particles with the plasmid containing the exogenous DNA.
  • the target cell can be surrounded by the plasmid so that the plasmid is carried into the cell by the wake of the particle.
  • the vector can be introduced in vivo by lipofection.
  • liposomes for encapsulation and transfection of nucleic acids in vitro.
  • Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et. al. Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987; Mackey, et al. Proc. Natl. Acad. Sci. USA 85:8027-8031, 1988; Ulmer et al.
  • cationic lipids can promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Feigner and Ringold Science 337:387-388, 1989, the disclosure of which is incorporated by reference herein in its entirety).
  • Particularly useful lipid compounds and compositions for transfer of nucleic acids are described in WO95/18863 and WO96/17823, and in U.S. Pat. No. 5,459,127, the disclosures of which are incorporated herein by reference in their entireties.
  • the nucleic acid constructs encoding any of the modified HIV-I Gag polypeptides described herein are viral vectors.
  • Methods for constructing and using viral vectors are known in the art ⁇ See e.g., Miller and Rosman, BioTeck, 7:980-990, 1992).
  • the viral vectors are replication defective, that is, they are unable to replicate autonomously in the target cell.
  • the genome of the replication defective viral vectors that are used within the scope of the present disclosure lack at least one region that is necessary for the replication of the virus in the infected cell. These regions can either be eliminated (in whole or in part), or be rendered non-functional by any technique known to a person skilled in the art. These techniques include the total removal, substitution (by other sequences, in particular by the inserted nucleic acid), partial deletion or addition of one or more bases to an essential (for replication) region. Such techniques can be performed in vitro (for example, on the isolated DNA).
  • DNA viral vectors commonly include attenuated or defective DNA viruses, including, but not limited to, herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), Moloney leukemia virus (MLV) and human immunodeficiency virus (HFV) and the like.
  • HSV herpes simplex virus
  • EBV Epstein Barr virus
  • AAV adenovirus
  • AAV adeno-associated virus
  • MMV Moloney leukemia virus
  • HBV human immunodeficiency virus
  • Defective viruses that entirely or almost entirely lack viral genes, are preferred, as defective virus is not infective after introduction into a cell.
  • Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells.
  • a specific tissue can be specifically targeted.
  • particular vectors include, but are not limited to, a defective herpes virus 1 (HSVl) vector (Kaplitt et al. MoI. Cell. Neurosci., 2:320-330, 1991, the disclosure of which is incorporated herein by reference in its entirety), defective herpes virus vector lacking a glycoprotein L gene ⁇ See for example, Patent Publication RD 371005 A the disclosure of which is incorporated herein by reference in its entirety), or other defective herpes virus vectors ⁇ See e.g., WO 94/21807; and WO 92/05263, the disclosures of which are incorporated herein by reference in their entireties); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al.
  • the vectors encoding any of the modified HIV-I Gag polypeptides described herein may be adenovirus vectors.
  • Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a nucleic acid of the disclosure to a variety of cell types.
  • Various serotypes of adenovirus exist.
  • adenoviruses of animal origin preference is given, within the scope of the present disclosure, to type 2, type 5 or type 26 human adenoviruses (Ad 2 or Ad 5), or adenoviruses of animal origin ⁇ See e.g., WO94/26914 and WO2006/020071 , the disclosures of which are incorporated herein by reference in their entireties).
  • Those adenoviruses of animal origin that can be used within the scope of the present disclosure include adenoviruses of canine, bovine, murine (e.g., Mavl, Beard et al.
  • the adenovirus of animal origin is a canine adenovirus, such as a CAV2 adenovirus (e.g. Manhattan or A26/61 strain (ATCC VR-800)).
  • the replication defective adenoviral vectors may include the ITRs, an encapsidation sequence and the polynucleotide sequence of interest.
  • at least the El region of the adenoviral vector is non-functional.
  • the deletion in the El region preferably extends from nucleotides 455 to 3329 in the sequence of the Ad5 adenovirus (Pvull-BgHl fragment) or 382 to 3446 (Hinfil-Sau3A fragment).
  • E3 region e.g., WO95/02697, the disclosure of which is incorporated herein by reference in its entirety
  • E2 region e.g., WO94/28938, the disclosure of which is incorporated herein by reference in its entirety
  • E4 region e.g., WO94/28152, WO94/12649 and WO95/02697, the disclosures of which are incorporated herein by reference in their entireties
  • the adenoviral vector has a deletion in the El region (Ad 1.0).
  • El -deleted adenoviruses are disclosed in EP 185,573, the contents of which are incorporated herein by reference.
  • the adenoviral vector has a deletion in the El and E4 regions (Ad 3.0).
  • Examples of E1/E4- deleted adenoviruses are disclosed in WO95/02697 and WO96/22378, the disclosures of which are incorporated herein by reference in their entireties.
  • the replication defective recombinant adenoviruses can be prepared by any technique known to the person skilled in the art (See e.g., Levrero et al. Gene 101:195, 1991 ; EP 185 573; and Graham EMBO J, 3:2917, 1984, the disclosures of which are incorporated herein by reference in their entireties).
  • they can be prepared by homologous recombination between an adenovirus and a plasmid, which includes, inter alia, the DNA sequence of interest.
  • the homologous recombination is accomplished following co-transfection of the adenovirus and plasmid into an appropriate cell line.
  • the cell line that is employed should preferably (i) be transformable by the elements to be used, and (ii) contain the sequences that are able to complement the part of the genome of the replication defective adenovirus, preferably in integrated form in order to avoid the risks of recombination.
  • Examples of cell lines that can be used are the human embryonic kidney cell line 293 (Graham et al. J. Gen. Virol.
  • a pharmaceutical composition including a nucleic acid construct encoding any of the modified HIV-I Gag polypeptides described herein is introduced into a subject prior to exposure to HIV to elicit a protective immune response.
  • the nucleic acid construct is a plasmids.
  • the dose range can be varied according to the physical, metabolic and immunological characteristics of the subject.
  • a dose of at least about 1 mg of DNA is administered.
  • 12 mg or less of DNA is administered.
  • a single dose can be at least about 2 mg, or at least about 3 mg, or at least about 4 mg of DNA.
  • a single dose does not exceed about 6 mg, or about 8 mg or about 10 mg of DNA.
  • a single dose, or multiple doses separated by a time interval can be administered to elicit an immune response against HIV-I .
  • two doses, or three doses, or four doses, or five doses, or six doses or more can be administered to a subject over a period of several weeks, several months or even several years, to optimize the immune response.
  • a "prime-boost" regimen may be utilized to administer any of the modified HIV-I Gag polypetides described herein.
  • a nucleic acid encoding the modified HIV-I Gag polypeptide is administered as a DNA vaccine prime followed by an adenoviral vector boost.
  • Prime-boost regimens have shown promise in non-human primate models of HIV infection. Such regimens have the potential for raising high levels of immune responses.
  • a "primer" composition including a modified HIV-I Gag polypeptide that is the same as a modified HIV-I Gag polypeptide encoded by an adenoviral vector of an adenoviral vector composition can be administered to a subject.
  • the primer composition can be administered at least about one week before the administration of the "booster" composition including one or more adenoviral vectors.
  • the nucleic acid "primer" encoding the modified HIV-I Gag polypeptide can be administered as part of a gene transfer vector or as naked DNA.
  • Any gene transfer vector can be employed in the primer composition, including, but not limited to, a plasmid, a retrovirus, an adeno-associated virus, a vaccine virus, a herpesvirus, or an adenovirus.
  • the transfer vector is a plasmid.
  • nucleic acids encoding any of the modified HIV-I Gag polypeptides described above can be used to prime an immune response against HIV-I, in combination with administration of a composition including an adenovirus vector encoding a modified HIV-I Gag polypeptide.
  • modified HIV-I Gag polypeptide encoded by the nucleic acid sequence of the boost composition often is the same as the modified HIV-I Gag polypeptide encoded by the nucleic acid constructs of the primer composition, in some embodiments it may be appropriate to use a primer composition comprising a nucleic acid sequence encoding a modified HIV-I Gag polypeptide that is different from modified HIV-I Gag polypeptide encoded by the adenoviral vector composition.
  • the primer composition is administered to the mammal to prime the immune response to HIV-I.
  • more than one dose of primer composition can be provided in any suitable timeframe (e.g., at least about 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks, 16 weeks, or more prior to boosting).
  • the primer composition is administered to the mammal at least three months (e.g., three, six, nine, twelve, or more months) before administration of the booster composition.
  • the primer composition is administered to the mammal at least about six months to about nine months before administration of the booster composition.
  • More than one dose of booster composition can be provided in any suitable timeframe to maintain immunity.
  • any route of administration can be used to deliver the adenoviral vector composition and/or the primer composition to the mammal. Indeed, although more than one route can be used to administer the adenoviral vector composition and/or the primer composition, a particular route can provide a more immediate and more effective reaction than another route. Most commonly, the adenoviral vector composition and/or the primer composition is administered via intramuscular injection. The adenoviral vector composition and/or the primer composition also can be applied or instilled into body cavities, absorbed through the skin (for example, via a transdermal patch), inhaled, ingested, topically applied to tissue, or administered parenterally via, for instance, intravenous, peritoneal, or intraarterial administration.
  • the adenoviral primer composition and/or the booster composition can be administered in or on a device that allows controlled or sustained release, such as a sponge, biocompatible meshwork, mechanical reservoir, or mechanical implant.
  • a device that allows controlled or sustained release such as a sponge, biocompatible meshwork, mechanical reservoir, or mechanical implant.
  • Implants see, e.g., U.S. Patent 5,443,505, the disclosure of which is incorporated herein by reference in its entirety
  • devices see, e.g., U.S. Patent 4,863,457, the disclosure of which is incorporated herein by reference in its entirety
  • an implantable device e.g., a mechanical reservoir or an implant or a device comprised of a polymeric composition
  • adenoviral vector composition and/or the primer composition also can be administered in the form of sustained-release formulations (see, e.g., U.S. Patent 5,378,475, the disclosure of which is incorporated herein by reference in its entirety) comprising, for example, gel foam, hyaluronic acid, gelatin, chondroitin sulfate, a polyphosphoester, such as bis-2-hydroxyethyl-terephthalate (BHET), and/or a polylactic-glycolic acid.
  • sustained-release formulations see, e.g., U.S. Patent 5,378,475, the disclosure of which is incorporated herein by reference in its entirety
  • sustained-release formulations comprising, for example, gel foam, hyaluronic acid, gelatin, chondroitin sulfate, a polyphosphoester, such as bis-2-hydroxyethyl-terephthalate (BHET), and/or a polylactic-glycolic acid.
  • BHET bis-2-
  • a booster composition can include a single dose of adenoviral vector comprising at least about 1x10 5 particles (which also is referred to as particle units) of adenoviral vector.
  • the dose preferably is at least about 1x10 6 particles (for example, about lxl0 6 -lxl0 12 particles), more preferably at least about 1x10 7 particles, more preferably at least about IxIO 8 particles (e.g., about lxl0 8 -lxl0 ⁇ particles or about IxIO 8 - 1x10 12 particles), and most preferably at least about 1x10 9 particles (e.g., about lxl0 9 -lxl0 10 particles or about lxl0 9 -lxl0 12 particles), or even at least about IxIO 10 particles (e.g., about lxl0 10 -lxl0 12 particles) of the adenoviral vector.
  • the dose comprises no more than about 1x10 14 particles, preferably no more than about 1x10 13 particles, even more preferably no more than about 1x10 12 particles, even more preferably no more than about IxIO 1 1 particles, and most preferably no more than about IxIO 10 particles (e.g., no more than about 1x10 9 particles).
  • the adenoviral vector composition can comprise a single dose of adenoviral vector comprising, for example, about 1x10 6 particle units (pu), 2x10 )(6 pu, 4xlO 6 pu, IxIO 7 pu, 2xlO 7 pu, 4xlO 7 pu, IxIO 8 pu, 2xlO 8 pu, 4xlO 8 pu, IxIO 9 pu, 2x10 ) ' 9 pu, 4xlO 9 pu, IxIO 10 pu, 2xlO 10 pu, 4xlO 10 pu, IxIO 11 pu, 2xl ⁇ " pu, 4xlO ⁇ pu, 1x10 ) 12 pu, 2xlO 12 pu, or 4xlO 12 pu of adenoviral vector.
  • adenoviral vector comprising, for example, about 1x10 6 particle units (pu), 2x10 )(6 pu, 4xlO 6 pu, IxIO 7 pu, 2xlO 7 pu, 4xlO 7 pu, IxIO 8 pu, 2
  • Methodology to detect T cell responsiveness to HIV peptides via ELISPOT is based upon modification of the method described in Helms, T., et al., Direct visualization of cytokine-producing recall antigen-specific CD4 memory T cells in healthy individuals and HIV patients. J Immunol, 2000. 164(7): p. 3723-32, the disclosure of which is incorporated herein by reference in its entirety. Analysis is performed using a commercially available ELISPOT Kit (BD Biosciences). PBMC are stimulated with the modified HIV-I Gag polypeptides overnight at 37 0 C in the replicate wells for at an optimized density of 2 x 10 5 cell/well for all stimulations other than SEB, which is conducted at 5 x 10 4 cell/well.
  • mice Six to 8-wk-old mice are immunized with a total of 15 ⁇ g of DNA encoding a modified HIV-I Gag polypeptide at each immunization.
  • the groups include mice injected with 15 ⁇ g of vector with no insert (Control) or those injected with 15 ⁇ g of the vector. All mice receive lOO ⁇ l DNA immunizations bilaterally in the muscle using needle and syringe at 4 timepoints with 2-week intervals. Blood is collected prior to immunizations and at specified timepoints for immune analysis. All animal experiments are reviewed and approved by the Animal Care and Use Committee, Vaccine Research Center (VRC), National Institute of Allergy and Infectious Diseases (http ://www. niaid ,nih. gov/yrc) and performed in accordance with all relevant federal and National Institutes of Health guidelines and regulations.
  • VRC Animal Care and Use Committee
  • Vaccine Research Center VRC
  • National Institute of Allergy and Infectious Diseases http ://www
  • the level of immunogenicity is assessed using the methods described herein. It will be appreciated that the methods above may be used to assess the immunogenicity of any of the modified HIV-I Gag polypeptides described herein.
  • Immunogenicity of modified HIV-I Gag polypeptides may be assessed in a primate model using the following prime-boost assay.
  • DNA vectors encoding the modified HIV-I Gag polypeptide being assessed in a suitable carrier are administered to adult cynomolgus monkeys. If desired, the vectors may be administered multiple times at desired time intervals. Control animals are administered carrier alone. For example, the monkeys may receive three immunizations of 8 mg total DNA vaccine intramuscularly at weeks 0, 4, and 8. Thereafter, recombinant adenovirus encoding the modified HIV-I Gag protein may be administered at a desired time. For control animals, carrier without adenovirus is administered.
  • the recombinant adenovirus may be administered at week 38 or week 24.
  • the recombinant adenovirus may be administered at any desired dosage.
  • the recombinant adenovirus may be administered at l ⁇ " PU. 026-03024).
  • ELISPOT assays are utilized to monitor the emergence of vaccine-elicited T cell immune responses.
  • 96-well multiscreen plates are coated overnight with 100 ⁇ l/well of 5 ⁇ g/ml anti-human IFN- ⁇ (B27; BD Pharmingen) in endotoxin-free Dulbecco's PBS (D-PBS).
  • D-PBS Dulbecco's PBS
  • the plates are then washed three times with D- PBS containing 0.25% Tween-20 (D-PBS/Tween), blocked for 2 h with D-PBS containing 5% FBS at 37 0 C, washed three times with D-PBS/Tween, rinsed with RPMI 1640 containing 10% FBS to remove the Tween-20, and incubated with modified HIV-I Gag polypeptide and 2 x 10 5 PBMC in triplicate in 100 ⁇ l reaction volumes. Following an 18h incubation at 37 0 C, the plates are washed nine times with D-PBS/Tween and once with distilled water.
  • the plates are then incubated with 2 ⁇ g/ml biotinylated rabbit anti-human IFN- ⁇ (Biosource) for 2 h at room temperature, washed six times with Coulter Wash (Beckman-Coulter), and incubated for 2.5 h with a 1 :500 dilution of streptavidin-AP (Southern Biotechnology). Following five washes with Coulter Wash and one with PBS, the plates are developed with NBT/BCIP chromogen (Pierce), stopped by washing with tap water, air dried, and read using an ELISPOT reader (Hitech Instruments). Spot-forming cells (SFC) per 10 6 PBMC are calculated.
  • Biosource Biosource
  • Plasmid DNA encoding the modified HIV-I Gag polypeptide to be administered is prepared from bacterial cell cultures containing an appropriate selection medium. Following growth of bacterial cells harboring the plasmid, the plasmid DNA is purified from cellular components.
  • Vaccines are prepared under cGMP conditions.
  • the vaccines meet lot release specifications prior to administration.
  • the DNA vaccine is manufactured at an appropriate dose in phosphate buffered saline (PBS). Vials are aseptically filled to the desired volume.
  • PBS phosphate buffered saline
  • the resulting DNA is administered via a desired route.
  • the DNA may be administered to the deltoid muscle using the Biojector 2000 ® Needle-Free Injection Management SystemTM.
  • the injection site is disinfected and the area allowed to dry completely.
  • the skin around the injection site is held firmly while the syringe is placed against the injection site at a 90° angle.
  • the actuator is pressed and the material is released into the muscle. Continue to hold firmly for 3 seconds.
  • BIOJECTOR 2000® utilizes sterile, single-use syringes for variable dose, up to 1.0 mL, medication administration.
  • the DNA composition agent is delivered under pressure by a compressed CO 2 gas cartridge that is stored inside the BIOJECTOR®.
  • CO 2 When the BIOJECTOR®' s actuator is depressed, CO 2 is released, causing the plunger to push the study agent out of the sterile syringe through the skin and into the underlying tissue.
  • the study agent is expelled through a micro-orifice at high velocity in a fraction of a second to pierce the skin.
  • the CO 2 does not come in contact with the injectate and the syringe design prevents any back splatter or contamination of the device by tissue from the subject.
  • Vaccination is repeated at a desired interval to achieve the desired level of immune response.
  • An immune response against HIV-I is primed by administering DNA encoding the modified HIV-I Gag polypeptide to a human.
  • the modified HIV- 1 Gag polypeptide itself may be administered.
  • the immune response After the immune response has been primed, it is boosted by administering a viral vector, such as an adenovirus vector.
  • the viral vector may be administered in multiple doses at desired time intervals.
  • An immune response sufficient to inhibit HIV-I infection or to reduce at least one symptom of HIV-I infection is generated.

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Abstract

L'invention concerne de polypeptides Gag de VIH-1 modifiés dans lesquels un ou plusieurs acides aminés dans le polypeptide Gag de VIH-1 ont été remplacés par un ou plusieurs acides aminés de la région correspondante dans la protéine Gag de VIS. Des procédés et compositions pour utiliser les polypeptides Gag de VIH-1 modifiés pour induire une réponse immunitaire sont également révélés.
PCT/US2008/073395 2007-08-17 2008-08-15 Utilisation de protéines gag vih/vis chimériques pour optimiser des réponses de lymphocytes t induites par vaccin contre le gag du vih WO2009026183A1 (fr)

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WO2010096561A1 (fr) * 2009-02-18 2010-08-26 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Protéines gag du vih/vis de synthèse et leurs utilisations
WO2011047316A1 (fr) * 2009-10-16 2011-04-21 The United States Of America, As Represented By The Secretary Department Of Health And Human Services Séquences d'acides nucléiques codant pour des protéines mosaïques extensibles du vih
WO2011043830A3 (fr) * 2009-10-09 2011-06-16 New York University Procédés, agents et peptides destinés à induire une réponse immunitaire innée dans la vaccination contre le vih
WO2012168480A1 (fr) * 2011-06-10 2012-12-13 Institut Curie Agents et procédés pour la production d'adjuvants non infectieux dérivés de capsides de vih
WO2013135615A1 (fr) 2012-03-12 2013-09-19 Crucell Holland B.V. Lots d'adénovirus recombinants ayant des extrémités terminales modifiées
US8932607B2 (en) 2012-03-12 2015-01-13 Crucell Holland B.V. Batches of recombinant adenovirus with altered terminal ends
WO2016146844A1 (fr) 2015-03-18 2016-09-22 Janssen Vaccines & Prevention B.V. Dosages pour systèmes d'expression de recombinaison
WO2016166088A1 (fr) 2015-04-14 2016-10-20 Janssen Vaccines & Prevention B.V. Adénovirus recombiné exprimant deux transgènes avec un promoteur bidirectionnel
WO2017220499A1 (fr) 2016-06-20 2017-12-28 Janssen Vaccines & Prevention B.V. Promoteur bidirectionnel puissant et équilibré
WO2018146205A1 (fr) 2017-02-09 2018-08-16 Janssen Vaccines & Prevention B.V. Promoteur puissant et court pour l'expression de gènes hétérologues
US11473105B2 (en) 2016-05-12 2022-10-18 Janssen Vaccines & Prevention B.V. Potent and balanced bidirectional promoter
EP4118098A4 (fr) * 2020-03-11 2024-06-12 The Trustees of The University of Pennsylvania Procédés et composition pour administration de gènes à l'aide d'une particule virale modifiée

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Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010096561A1 (fr) * 2009-02-18 2010-08-26 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Protéines gag du vih/vis de synthèse et leurs utilisations
US9358281B2 (en) 2009-10-09 2016-06-07 New York University Methods, agents and peptides for inducing an innate immune response in HIV vaccination
WO2011043830A3 (fr) * 2009-10-09 2011-06-16 New York University Procédés, agents et peptides destinés à induire une réponse immunitaire innée dans la vaccination contre le vih
EP2485759A2 (fr) * 2009-10-09 2012-08-15 New York University Procédés, agents et peptides destinés à induire une réponse immunitaire innée dans la vaccination contre le vih
EP2485759A4 (fr) * 2009-10-09 2013-07-24 Univ New York Procédés, agents et peptides destinés à induire une réponse immunitaire innée dans la vaccination contre le vih
WO2011047316A1 (fr) * 2009-10-16 2011-04-21 The United States Of America, As Represented By The Secretary Department Of Health And Human Services Séquences d'acides nucléiques codant pour des protéines mosaïques extensibles du vih
WO2012168480A1 (fr) * 2011-06-10 2012-12-13 Institut Curie Agents et procédés pour la production d'adjuvants non infectieux dérivés de capsides de vih
US8932607B2 (en) 2012-03-12 2015-01-13 Crucell Holland B.V. Batches of recombinant adenovirus with altered terminal ends
WO2013135615A1 (fr) 2012-03-12 2013-09-19 Crucell Holland B.V. Lots d'adénovirus recombinants ayant des extrémités terminales modifiées
WO2016146844A1 (fr) 2015-03-18 2016-09-22 Janssen Vaccines & Prevention B.V. Dosages pour systèmes d'expression de recombinaison
WO2016166088A1 (fr) 2015-04-14 2016-10-20 Janssen Vaccines & Prevention B.V. Adénovirus recombiné exprimant deux transgènes avec un promoteur bidirectionnel
US10570417B2 (en) 2015-04-14 2020-02-25 Janssen Vaccines & Prevention B.V. Recombinant adenovirus expressing two transgenes with a bidirectional promoter
US11473105B2 (en) 2016-05-12 2022-10-18 Janssen Vaccines & Prevention B.V. Potent and balanced bidirectional promoter
WO2017220499A1 (fr) 2016-06-20 2017-12-28 Janssen Vaccines & Prevention B.V. Promoteur bidirectionnel puissant et équilibré
US11001858B2 (en) 2016-06-20 2021-05-11 Janssen Vaccines & Prevention B.V. Potent and balanced bidirectional promoter
US11781155B2 (en) 2016-06-20 2023-10-10 Janssen Vaccines & Prevention B.V. Potent and balanced bidirectional promoter
WO2018146205A1 (fr) 2017-02-09 2018-08-16 Janssen Vaccines & Prevention B.V. Promoteur puissant et court pour l'expression de gènes hétérologues
US11034978B2 (en) 2017-02-09 2021-06-15 Janssen Vaccines & Prevention B.V. Potent and short promoter for expression of heterologous genes
EP4118098A4 (fr) * 2020-03-11 2024-06-12 The Trustees of The University of Pennsylvania Procédés et composition pour administration de gènes à l'aide d'une particule virale modifiée

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