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CD 4 T细胞组成。新出现的信息表明，这些细胞由于两个不同的原因内在地抵抗凋亡：（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抗性表型，和（ii）静息记忆T细胞，由于是先前免疫应答的历史档案，其发展为静止和抗凋亡状态，以保存记忆应答。 目前“治愈”HIV的方法广泛涉及基因治疗、基于免疫的治疗和病毒再激活。后一种策略涉及重新激活HIV-1，预期重新激活病毒的CD 4 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病毒再活化是可能的（例如，与辛二酰苯胺异羟肟酸，SAHA）和安全，但考虑到这些细胞对凋亡的内在抗性，再活化病毒的细胞在再活化后既不死亡，也不被细胞毒性T淋巴细胞有效杀死，这并不奇怪。 等表征了含有潜伏HIV的静息记忆CD 4 T细胞中选择的凋亡调节蛋白的表达，并发现细胞具有低水平的促凋亡蛋白半胱氨酸天冬氨酸蛋白酶原8和高水平的抗凋亡蛋白Bcl 2。我们认为，这种不平衡是潜伏性HIV感染的CD 4 T细胞在HIV再活化后不死亡的原因，尽管事实上它们在细胞内表达有效的凋亡诱导蛋白- HIV达特、nef、Vpr和蛋白酶。因此，潜伏感染的细胞 甚至在诱导表达促凋亡HIV蛋白如HIV蛋白酶后也不死亡。 这项研究的总体目标是确定改变潜伏感染的HIV T细胞的方法，使它们在病毒重新激活时死亡。在本申请中，我们提出了三条独立的证据，证明这种方法是合理的，这些细胞可以被改变，当HIV被重新激活时，这些细胞就会死亡。首先使用Lewin模型的HIV潜伏期在初级CD 4 T细胞中，我们表明，上调宿主蛋白procaspase 8，在静息记忆CD 4 T细胞中，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HIV拷贝。接下来，我们总结了我们以前发表的工作，即用TRAIL激动剂治疗来自HIV感染患者的静息记忆CD 4 T细胞减少了复制能力HIV的量和HIV前病毒的量，而对未感染的旁观者细胞没有有害影响。最后，我们提出的初步证据表明，第一类Bcl 2抑制剂，ABT-737，引发潜伏感染的细胞进行死亡后，艾滋病毒重新激活。这些方法特异性地靶向HIV感染的细胞死亡，因为使用这种策略，所有细胞将被引发成为凋亡敏感的，然而，只有那些含有细胞内HIV蛋白的细胞（HIV感染的细胞）含有凋亡诱导刺激物。 已经证明了我们的HIV根除的“Prime Shock and Kill”模型的概念，我们现在提出采用高通量筛选方法来鉴定i）诱导凋亡敏感性的最佳药理学方法，然后ii）测试这些治疗与诱导病毒再活化的刺激物的组合。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感染患者的原代静息CD 4 T细胞的离体测试引起潜伏HIV感染的T细胞死亡。最终成功的方法将使用BLT小鼠HIV感染模型进行全面审查。