Prime shock and kill for HIV erradication

为根除艾滋病毒提供休克和杀戮

• 批准号: 8657290
• 负责人: ANDREW D BADLEY
• 金额: $ 55.51万
• 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/8657290
• 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; 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; pro-caspase-8; protein expression; public health relevance; 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的主要宿主是潜伏感染的静止记忆CD4T细胞。新的信息表明，这些细胞对凋亡具有内在的抵抗力，原因有两个：(I)慢性HIV感染T细胞通过HIV蛋白引起多种凋亡调节蛋白的表达变化而诱导抗凋亡表型；(Ii)静止的记忆T细胞，由于其是先前免疫反应的历史档案，形成静止的和抗凋亡的状态以保存记忆反应。目前“治愈”艾滋病毒的方法广泛地包括基因疗法、基于免疫的疗法和病毒复活。后一种策略包括从药物上重新激活HIV，期望重新激活病毒的CD4T细胞将死于病毒蛋白表达的细胞毒性效应。到目前为止的工作已经证明，病毒重新激活是可能的(例如，使用异羟肟酸，SAHA)和安全的，但考虑到这些细胞对凋亡的内在抵抗力，难怪使病毒重新激活的细胞在重新激活后既不会死亡，也不会被细胞毒性T淋巴细胞有效地杀死。他们对含有潜伏的HIV的静止记忆CD4T细胞中部分凋亡调节蛋白的表达进行了表征，发现这些细胞具有低水平的促凋亡蛋白proaspase8和高水平的抗凋亡蛋白Bcl2。我们认为，这种不平衡是潜伏感染HIV的CD4T细胞在HIV重新激活后没有死亡的原因，尽管它们在病毒重新激活后在细胞内表达强大的凋亡诱导蛋白-HIV TAT、NEF、VPR和蛋白酶。因此，潜伏感染的细胞 即使在被诱导表达促凋亡的艾滋病毒蛋白(如艾滋病毒蛋白酶)后，它们也不会死亡。这项拟议研究的首要目标是确定改变潜伏感染的艾滋病毒T细胞的方法，使它们在病毒重新激活时死亡。在这项申请中，我们提出了三条独立的证据，证明这种方法是合理的，这些细胞可以通过这样的方式改变，当HIV重新激活时，这些细胞将死亡。首先使用原代CD4T细胞中HIV潜伏期的勒文模型，我们证明在药物上上调静止记忆CD4T细胞中的宿主蛋白proaspase8，允许这些细胞在病毒重新激活后被杀死，导致较低的HIV复制(因为感染的细胞被杀死)和较少的整合的HIV拷贝。接下来，我们总结了我们之前发表的工作，即用TRAIL激动剂治疗来自HIV感染患者的静止记忆CD4T细胞减少了具有复制能力的HIV的数量和HIV前病毒的数量，而不会对未感染的旁观者细胞产生有害影响。最后，我们提出了初步证据，第一类Bcl2抑制剂，ABT-737，启动潜伏感染的细胞经历艾滋病毒重新激活后死亡。这些方法专门针对HIV感染的细胞死亡，因为使用这种策略，所有的细胞都将准备变得对凋亡敏感，然而，只有那些含有细胞内HIV蛋白的细胞(HIV感染的细胞)才含有诱导凋亡的刺激。在证明了我们根除HIV的“主要休克和杀死”模式的概念后，我们现在建议采用高通量筛选方法来确定最佳药理学方法，即i)诱导细胞凋亡敏感性，然后，ii)结合诱导病毒重新激活的刺激来测试这些治疗方法。然后，将测试这种方法是否有能力 使用HIV潜伏期的体外模型和来自HIV感染患者的主要静止的CD4T细胞的体外测试来导致潜伏的HIV感染的T细胞死亡。最终，将使用艾滋病毒感染的BLT小鼠模型对成功的方法进行全面审查。