Prime shock and kill for HIV erradication

为根除艾滋病毒提供休克和杀戮

• 批准号: 8996120
• 负责人: ANDREW D BADLEY
• 金额: $ 69.02万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-15 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8996120
• 关键词: Acute; Adopted; Agonist; Anti-Retroviral Agents; Apoptosis; Archives; CD4 Positive T Lymphocytes; Cause of Death; Cell Death; Cell model; Cell physiology; Cells; Cessation of life; Chemosensitization; Chronic; Communicable Diseases; Communities; Cytotoxic T-Lymphocytes; Disease; Equilibrium; Exhibits; Foundations; Goals; HIV; HIV Infections; HIV Protease; HIV tat Protein; HIV therapy; Health; Immune; Immune response; Infection; Infectious Agent; Lead; Left; Life; Malignant Neoplasms; Memory; Methods; Modeling; Modification; Myeloablative Chemotherapy; Patients; Peptide Hydrolases; Persons; Pharmacologic Substance; Phenotype; Predisposition; Production; Proteins; Proviruses; Publishing; Resistance; Rest; Sampling; Shock; Signal Transduction; Stem cell transplant; Stimulus; T memory cell; T-Lymphocyte; TNFSF10 gene; Testing; Time; Toxic effect; Vaccines; Viral; Viral Proteins; Virus; Vorinostat; Work; base; cancer cell; cell killing; chemotherapy; clinical application; cytotoxic; expectation; gene therapy; genetic regulatory protein; high throughput screening; immune activation; in vitro Model; inhibitor/antagonist; killings; macrophage; memory CD4 T lymphocyte; mouse model; neoplastic cell; novel; novel strategies; prevent; protein expression; research study; response; therapy design

DESCRIPTION: The primary reservoir of HIV consists of latently infected resting memory CD4 T cells. Emerging information indicates that these cells are intrinsically resistant to apoptosis fr two distinct reasons: (i) chronic HIV infection of T cells induces an apoptosis resistant phenotype by virtue of HIV proteins causing altered expression of a wide variety of apoptosis regulatory proteins, and (ii) resting memory T cells, by virtue of being an historical archive of prior immune responses developing a quiescent and apoptosis resistant state in order to preserve the memory responses. Current approaches to "cure" HIV broadly involve gene therapy, immune based therapy, and viral reactivation. The latter strategy involves reactivating HIV pharmacologically, with the expectation that CD4 T cells which reactivate virus will die from the cytotoxic effects of viral protein expression. Work to date has established that viral reactivation is possible (e.g., with suberoylanilide hydroxamic acid, SAHA) and safe, but given the intrinsic resistance of these cells to apoptosis, it is not surprising that the cells that reacivate virus neither die after reactivation, nor are they efficiently killed by cytotoxic T lymphocytes. e have characterized the expression of select apoptosis regulatory proteins in resting memory CD4 T cells which contain latent HIV, and found the cells to have low levels of the proapoptotic protein procaspase 8 and high levels of the antiapoptotic protein Bcl2. We propose that this imbalance is the reason why latently HIV infected CD4 T cells do not die after HIV reactivation, despite the fact that they express potent apoptosis-inducing proteins intracellularly - HIV Tat, nef, Vpr and protease after viral reactivation. Therefore, the cells that were latently infected do not die even after they are induced to express proapoptotic HIV proteins such as HIV protease. The overarching goal of the proposed study is to identify ways to alter latently infected HIV T cells such that they die in response to viral reactivation. In this application, we present three independent lines of evidence that this approach is justified and these cells can be altered in such a way that when HIV is reactivated, the cells will die. First using the Lewin model of HIV latency in primary CD4 T cells, we show that pharmacologically up-regulating the host protein procaspase 8, in resting memory CD4 T cells, allows these cells to be killed after viral reactivation, resulting in lower HIV replication (because infected cells are killed) and less integrated HIV copies. Next we summarize our previously published work that treatment of resting memory CD4 T cells from HIV infected patients with TRAIL agonists reduces that amount of replication competent HIV and the amount of HIV provirus, without deleterious effects on uninfected bystander cells. Finally, we present preliminary evidence that the first in class Bcl2 inhibitor, ABT-737, primes latently infected cells to undergo death upon HIV reactivation. These approaches specifically target HIV infected cells to die because, using this tactic, all cell will be primed to become apoptosis susceptible, however, only those cells which contain intracellular HIV proteins (the HIV infected cells) contain the apoptosis inducing stimulus. Having shown proof of concept for our "Prime Shock and Kill" model of HIV eradication, we now propose to adopt a high throughput screening approach to identify optimum pharmacologic methods of i) inducing apoptosis sensitivity, and then, ii) test these treatments in combination with stimuli that induce viral reactivation. This approach will then be tested for their ability to cause latently HIV infected T cell death using in vitro models of HIV latency and ex vivo testing of primary resting CD4 T cells from HIV-infected patients. Ultimately successful approaches will be fully vetted using the BLT mouse model of HIV infection.

描述：HIV的主要储层由潜在感染的静止记忆CD4 T细胞组成。新出现的信息表明，这些细胞在本质上对凋亡具有两个不同的原因：（i）T细胞的慢性HIV感染诱导hiv蛋白诱导凋亡的表型，从而改变了各种凋亡调节蛋白的表达，从而改变了多种凋亡调节蛋白的表达，并通过历史上的育儿构成的戒烟，（ii）在历史抗性的情况下，（ii）避免了戒烟，（ii为了保留内存响应。 当前的“治愈” HIV的方法广泛涉及基因治疗，基于免疫治疗和病毒重新激活。后一种策略涉及在药理学上重新激活HIV，预计将病毒重新激活的CD4 T细胞将死于病毒蛋白表达的细胞毒性作用。迄今为止的工作已经确定了病毒重新激活是可能的（例如，使用suberoylanilide羟花酸，saha）且安全，但是鉴于这些细胞对凋亡的固有耐药性，毫不奇怪，重新启动病毒的细胞既不是在重新激活后也没有死亡，也没有被细胞毒素毒性毒性毒性杀死。 E表征了含有潜在HIV的静息记忆CD4 T细胞中选择凋亡调节蛋白的表达，并发现细胞具有较低的促凋亡蛋白procaspase 8和高水平的抗凋亡蛋白BCL2。我们认为，这种失衡是艾滋病毒重新激活后未受潜在的HIV感染CD4 T细胞死亡的原因，尽管它们在病毒重生后会内细胞内表达有效的凋亡诱导蛋白质 - HIV TAT，NEF，VPR和蛋白酶。因此，被潜在感染的细胞DO 即使诱使它们表达促凋亡的HIV蛋白（例如HIV蛋白酶），也不会死亡。 拟议的研究的总体目标是确定改变潜在感染的HIV T细胞的方法，使它们因病毒重新激活而死亡。在此应用中，我们提出了三种独立的证据，表明该方法是合理的，并且可以改变这些细胞，以至于当HIV重新激活时，细胞会死亡。首先使用原代CD4 T细胞中的HIV潜伏期LEWIN模型，我们表明，在静止的记忆CD4 T细胞中，在药理学上上调了宿主蛋白procaspase 8的药理上调，使这些细胞在病毒重新激活后被杀死，导致HIV复制较低（因为受感染的细胞被杀死）和较少的综合HIV拷贝。接下来，我们总结了我们先前发表的工作，即受HIV感染的静息记忆CD4 T细胞的治疗减少了这种复制量的竞技HIV和HIV病毒量，而对未感染的信托手细胞没有有害影响。最后，我们提供了初步证据表明，BCl2类抑制剂ABT-737中的第一个prime被刺激性细胞受到艾滋病毒重新激活后死亡。这些方法专门针对受艾滋病毒感染的细胞死亡，因为使用这种策略，所有细胞都将被启发为凋亡易感性，但是，只有那些含有细胞内HIV蛋白（HIV感染细胞）的细胞含有诱导刺激的细胞凋亡。 在我们为消除HIV的“主要冲击和杀死”模型的概念验证后，我们现在建议采用高吞吐量筛选方法来识别i）i）i）诱导凋亡敏感性的最佳药理方法，然后，ii）与刺激结合诱导病毒性重新激活。然后，将对这种方法的能力进行测试 使用HIV潜伏期的体外模型和对HIV感染患者的原发性静息CD4 T细胞的体外模型引起潜在的HIV感染T细胞死亡。最终，使用HIV感染的BLT小鼠模型将对成功的方法进行全面审查。