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 的主要储存库由潜伏感染的静息记忆 CD4 T 细胞组成。新出现的信息表明，这些细胞本质上对细胞凋亡具有抵抗力，原因有两个：（i）T细胞的慢性HIV感染通过HIV蛋白引起多种细胞凋亡调节蛋白的表达改变而诱导细胞凋亡抵抗表型，以及（ii） ）静息记忆 T 细胞，由于是先前免疫反应的历史档案，形成静止和抗凋亡状态，以保留记忆反应。 目前“治愈”艾滋病毒的方法广泛涉及基因疗法、基于免疫的疗法和病毒再激活。后一种策略涉及在药理学上重新激活HIV，期望重新激活病毒的CD4 T细胞将因病毒蛋白表达的细胞毒性作用而死亡。迄今为止的工作已经证实病毒重新激活是可能的（例如，使用辛二酰苯胺异羟肟酸，SAHA）并且是安全的，但考虑到这些细胞对细胞凋亡的内在抵抗力，重新激活病毒的细胞在重新激活后既不死亡也不奇怪也就不足为奇了。它们是否被细胞毒性 T 淋巴细胞有效杀死？ 我们对含有潜伏 HIV 的静息记忆 CD4 T 细胞中选定的凋亡调节蛋白的表达进行了表征，并发现这些细胞具有低水平的促凋亡蛋白 procaspase 8 和高水平的抗凋亡蛋白 Bcl2。我们认为，这种不平衡是潜伏感染 HIV 的 CD4 T 细胞在 HIV 重新激活后不会死亡的原因，尽管它们在病毒重新激活后在细胞内表达有效的细胞凋亡诱导蛋白 - HIV Tat、nef、Vpr 和蛋白酶。因此，被潜伏感染的细胞 即使它们被诱导表达促凋亡 HIV 蛋白（例如 HIV 蛋白酶）后也不会死亡。 拟议研究的总体目标是找到改变潜伏感染的 HIV T 细胞的方法，使它们因病毒重新激活而死亡。在本申请中，我们提出了三个独立的证据，证明这种方法是合理的，并且这些细胞可以被改变，使得当艾滋病毒重新激活时，细胞将会死亡。首先使用原代 CD4 T 细胞中 HIV 潜伏期的 Lewin 模型，我们发现通过药理学上调静息记忆 CD4 T 细胞中的宿主蛋白 procaspase 8，可以使这些细胞在病毒重新激活后被杀死，从而导致 HIV 复制降低。因为受感染的细胞被杀死）并且 HIV 拷贝的整合程度较低。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 HIV 感染患者的静息记忆 CD4 T 细胞，减少了具有复制能力的 HIV 数量和 HIV 原病毒的数量，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，表明首个 Bcl2 抑制剂 ABT-737 可在 HIV 重新激活时引发潜伏感染细胞死亡。这些方法专门针对HIV感染细胞进行死亡，因为使用这种策略，所有细胞都将变得容易凋亡，然而，只有那些含有细胞内HIV蛋白的细胞（HIV感染细胞）才含有诱导凋亡的刺激物。 在展示了我们的 HIV 根除“Prime Shock and Kill”模型的概念证明后，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：i) 诱导细胞凋亡敏感性，然后 ii) 组合测试这些治疗方法用诱导病毒重新激活的刺激。然后将测试这种方法的能力 使用 HIV 潜伏期体外模型和对 HIV 感染患者的原代静息 CD4 T 细胞进行离体测试，导致潜伏 HIV 感染的 T 细胞死亡。最终成功的方法将使用 HIV 感染的 BLT 小鼠模型进行全面审查。