Determining and Overcoming barriers in translating HIV-1 protease mediated activation of the CARD8 inflammasome to HIV cure research

确定并克服将 HIV-1 蛋白酶介导的 CARD8 炎性体激活转化为 HIV 治愈研究的障碍

• 批准号: 10749872
• 负责人: Kolin Clark
• 金额: $ 2.12万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2023-10-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10749872
• 关键词: Antigens; Apoptosis; Biological Assay; CASP1 gene; CD4 Positive T Lymphocytes; Caspase; Cell Death; Cells; Cessation of life; Chemicals; Chronic; Clinic; Clinical; Dimerization; Dipeptidyl Peptidases; Epidemic; Family member; Genes; Genetic Variation; Genetic study; HIV; HIV Genome; HIV Infections; HIV envelope protein; HIV therapy; HIV-1; HIV-1 protease; Immune; Immune response; In Vitro; Infection; Inflammasome; Inflammatory; Integration Host Factors; Investigation; Left; Mediating; Methods; NNRTI-resistance; Nature; Pathway interactions; Patients; Pattern recognition receptor; Peptide Hydrolases; Persons; Pharmaceutical Preparations; Process; Protein Isoforms; Proteins; RNA-Directed DNA Polymerase; Research; Resistance; Role; Sampling; Shock; Surface; T-Cell Activation; Testing; Translating; Variant; Viral; Viral reservoir; Virion; Virus; Virus Latency; Work; adaptive immune response; antiretroviral therapy; cell type; cellular targeting; clinically relevant; efavirenz; experimental study; genetic association; genetic variant; genome wide association study; global health; humanized mouse; in vivo; inhibitor; mouse model; non-nucleoside reverse transcriptase inhibitors; novel; pol genes; premature; prevent; recruit; resistance mechanism; translatable strategy; treatment strategy; virus genetics; whole genome

Project Summary/Abstract Human immunodeficiency virus (HIV) has maintained its status as a global epidemic since the 1980’s and 38 million people are living with HIV globally. While new HIV infections have been decreasing and antiretroviral therapies (ART) can maintain viral suppression in PLWH, there is no effective cure for elimination of the virus from the body. HIV is adept in escaping the normal immune response through the seeding of viral latent reservoirs primarily in quiescent CD4+ T cells. Upon halting ART these latent reservoirs are capable of spreading infection. In addition to this hurdle, HIV is able to escape the adaptive immune response due to the reliance upon recognition of the highly mutable HIV envelope. Therefore, it is critical to develop a cure strategy that can sense a highly immutable aspect of HIV such as those with essential enzymatic activity. Our previous work has shown that HIV-1 protease is one such protein that can be a target for a cure strategy. The pattern recognition receptor caspase recruitment domain-contain protein 8 (CARD8) is able to sense HIV-1 protease and activate pyroptosis, an inflammatory version of programmed cell death. This strategy requires the premature intracellular activation of the protease which can be achieved through the use of non-nucleoside reverse transcriptase inhibitors (NNRTIs) Efavirenz or Rilpivirine. Upon addition of NNRTIs to HIV infected cells, CARD8 activates the inflammasome and induces pyroptosis. Additionally, my previous work showed that inhibition of the negative regulator of dipeptidyl peptidase 9 (DPP9) is able to enhance NNRTI-mediated pyroptosis. However, during the discovery of this method for viral reservoir clearance, we found significant variation in the activation of the CARD8 inflammasome that could be ascribed to both the virus and the host. In fact, pyroptosis efficiency varied across viral strains, CARD8 isoforms, cell types, CD4+ T cell activation state, and CD4+ T cell donors. Therefore, the central hypothesis of this study is that there are genetic variants in both the virus and the host that can confer resistance to NNRTI induced HIV-1 protease activation of the CARD8 inflammasome and which may be overcome by CARD8 inflammasome sensitization through DPP9 inhibition. I will first test clinical and in vivo applications of CARD8 enhancement through DPP9 inhibition in Aim 1 by testing clinical isolates and enhancement in a humanized mouse model. Experiments in Aim 2 will identify the critical viral components needed for CARD8 sensing of HIV-1 protease activity to inform a genome wide association study of HIV-1 to identify variants responsible for pyroptosis variation. Aim 3 will elucidate host factors that may cause variation in protease sensing or inflammasome activation. This will be ascertained through the study of isoform expression in the key cellular targets for HIV-1 infection, through the identification of donors with varying killing efficiency, and through potentially identifying novel negative regulators of CARD8. Collectively, these studies will describe the potential efficacy of NNRTI induced CARD8 inflammasome activation as a viral reservoir reduction strategy and is the first step in translating this work to the clinic.

项目摘要/摘要 自1980年代以来，人类免疫缺陷病毒（HIV）一直保持其作为全球流行病的地位和38 百万人在全球享有艾滋病毒。虽然新的HIV感染正在减少和抗逆转录病毒 疗法（ART）可以在PLWH中维持病毒抑制，没有有效的治疗方法可以消除病毒 从身体。 HIV适应通过病毒潜伏的播种来逃避正常的免疫反应 静态CD4+ T细胞中的储层主要。停止艺术时，这些潜在水库能够 传播感染。除了这个障碍之外，艾滋病毒还能够逃脱由于 依赖对高度可变的HIV信封的认可。因此，制定治愈策略至关重要 这种感觉可以感觉到HIV的高度不变方面，例如具有必不可少的酶活性的艾滋病毒。我们的先前 工作表明，HIV-1蛋白是一种可以成为治愈策略的靶标的蛋白质。图案 识别受体caspase募集结构域蛋白8（card8）能够感知HIV-1蛋白酶 并激活凋亡，这是程序性细胞死亡的炎症版本。此策略需要 蛋白酶的过早细胞内激活可以通过使用非核苷来实现 逆转录酶抑制剂（NNRTIS）efavirenz或rilpivirine。在添加NNRTIS后感染了HIV 细胞，Card8激活炎症体并诱导凋亡。此外，我以前的工作表明 抑制二肽基肽酶9（DPP9）的阴性调节剂能够增强NNRTI介导 凋亡。但是，在发现这种病毒储层清除方法的过程中，我们发现了重要的 可以分配给病毒和宿主的Card8炎症体激活的变化。 实际上，凋亡效率在病毒菌株，Card8同工型，细胞类型，CD4+ T细胞激活之间变化 状态和CD4+ T细胞供体。因此，这项研究的中心假设是有遗传变异 在病毒和宿主中，可以赋予NNRTI抗性的HIV-1蛋白酶激活 CARD8炎症体，可以通过DPP9通过Card8炎性敏感性来克服它 抑制。我将首先测试通过DPP9抑制Card8增强的临床和体内应用 AIM 1通过测试人源化小鼠模型中的临床分离株和增强。 AIM 2中的实验将 确定HIV-1蛋白酶活性的CARD8感知所需的关键病毒成分，以告知基因组 HIV-1的广泛关联研究，以鉴定负责凋亡变异的变体。 AIM 3将阐明 可能导致蛋白酶传感或炎症体激活变化的宿主因子。这将被确定 通过研究HIV-1感染的关键细胞中的同工型表达，通过鉴定 通过具有不同杀伤效率的捐助者，并通过潜在地识别CARD8的新型负调节剂。 总的来说，这些研究将描述NNRTI诱导的Card8炎症体的潜在有效性 激活作为病毒的减少策略，是将这项工作转化为诊所的第一步。