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    • 4. 发明申请
    • CONDITIONALLY CONTROLLED, ATTENUATED HIV VACCINE
    • 定期控制,减毒的艾滋病毒疫苗
    • WO0107637B1
    • 2001-11-22
    • PCT/US0040478
    • 2000-07-25
    • SMITH STEPHEN
    • SMITH STEPHEN
    • C12N15/09A61K31/65A61K35/12A61K39/21A61K48/00A61P31/18A61P43/00C12N5/10C12N7/04C12N15/85C12N15/867C12N15/86
    • C12N15/86A61K39/12A61K39/21A61K2039/5254C12N7/00C12N15/85C12N2740/16022C12N2740/16034C12N2740/16043C12N2740/16051C12N2830/003
    • The present invention provides an attenuated HIV vaccine comprising an HIV virus modified to replicate only in the presence of at least one tetracycline analogue. Additionally, the present invention provides a method for immunization of humans against HIV which comprises administering to a human a vaccine including an HIV virus modified to replicate only in the presence of at least one tetracycline analogue. Simultaneously, at least one tetracycline analogue is administered for a period of time to allow replication of the modified HIV virus in vivo sufficient to produce immunity. Preferably, the tetracycline analogue is doxycycline. A replication competent HIV-DoxT virus genome which can be controlled by the presence or absence of doxycycline is produced by preparing a promoter, TetopTCAT; producing a provirus, pHIV-DoxT, using the TetopCAT promoter, and transfecting the pHIV-DoxT in cell lines in the presence of doxycycline. A replication competent HIV-DoxSp virus genome which can be controlled by the presence or absence of doxycycline is produced by preparing a promoter, TetopSpCAT; producing a provirus, pHIV-DoxSp, using the TetopCAT promoter, and transfecting the pHIV-DoxSp in cell lines in the presence of doxycycline. Anti-HIV vaccines are prepared using the HIV-DoxT and HIV-DoxSp virus genomes. These viruses and doxycycline are administered to human hosts. The doxycycline is administered for a time sufficient to build up immunity and then the administration of the drug is stopped so that the doxycycline-dependent viruses will no longer replicate.
    • 本发明提供了减毒的HIV疫苗,其包含仅在存在至少一种四环素类似物的情况下才被复制的HIV病毒。 此外,本发明提供了一种免疫人体抗HIV的方法,该方法包括给人施用包括仅在至少一种四环素类似物存在下才能复制的HIV病毒的疫苗。 同时,给予至少一种四环素类似物一段时间以使修饰的HIV病毒体内复制足以产生免疫力。 优选地,四环素类似物是多西环素。 可通过存在或不存在强力霉素来控制的具有复制能力的HIV-DoxT病毒基因组通过制备启动子TetopTCAT产生; 使用TetopCAT启动子产生前病毒,pHIV-DoxT,并在多西环素存在下在细胞系中转染pHIV-DoxT。 可以通过存在或不存在强力霉素来控制的复制型HIV-DoxSp病毒基因组通过制备启动子TetopSpCAT产生; 使用TetopCAT启动子产生前病毒,pHIV-DoxSp,并在多西环素存在下在细胞系中转染pHIV-DoxSp。 使用HIV-DoxT和HIV-DoxSp病毒基因组制备抗HIV疫苗。 这些病毒和强力霉素被施用于人类宿主。 施用强力霉素足够长的时间以建立免疫性,然后停止施用药物,以使强力霉素依赖性病毒不再复制。
    • 8. 发明申请
    • Conditionally controlled, attenuated HIV vaccine
    • 有条件控制,减毒的艾滋病毒疫苗
    • WO0107637A3
    • 2001-08-23
    • PCT/US0040478
    • 2000-07-25
    • SMITH STEPHEN
    • SMITH STEPHEN
    • C12N15/09A61K31/65A61K35/12A61K39/21A61K48/00A61P31/18A61P43/00C12N5/10C12N7/04C12N15/85C12N15/867C12N15/86
    • C12N15/86A61K39/12A61K39/21A61K2039/5254C12N7/00C12N15/85C12N2740/16022C12N2740/16034C12N2740/16043C12N2740/16051C12N2830/003
    • A live attenuated human immunodeficiency virus type 1 (HIV-1) whose replication is not constitutive but is instead conditionally regulated (such that rounds of reverse transcription with accompanying potential for error are strictly limited) might yield a paradigm that minimizes evolution to virulence and facilitate vaccine development. We have broached the concept of conditional control of HIV-1 through gain-of-function. Here, we describe the design of constitutively inactive HIV-1 genomes (HIV-DoxT and HIV-DoxSp) which can be conditionally resuscitated to an active state by tetracycline or related analogues. The HIV-DoxT construct comprises an inactivating mutation engineered into TAR, thereby rendering the virus non-responsive to Tat, a 302-bp DNA fragment (TetopT) which contains the tet-operator ligated into a position upstream of the HIV TATAA box, in both the 5' and 3' LTRs, and a reverse tetracycline-controlled activator (RTTA) coding sequence in place of the nef coding region. The HIV-DoxSp construct contains three additional Sp1 sites in the TetopT promoter upstream of the TATAA box thereby generating the promoter TetopSp. Genotypically, HIVDoxT is tat(+)tar(-)nef(-)Sp1(-) and HIVDoxSp is tat(+)tar(-)nef(-)Sp1(+). Since both genomes are genetically tar(-), they would ordinarily be expected to be wholly defective in producing viral proteins and/or particles. However, following transfection into an appropriate cell target, both proviruses, in a doxycycline-dependent fashion, capably released Gag and RT from cells. In the absence of doxycycline, no replication competent virus could be recovered. These findings suggest that the heterologous RTTA+Dox mechanism substituted effectively for Tat/TAR. These constructs should prove useful in the development of HIV-specific immunological and diagnostic reagents.