Defining HIV Env protein expression in latently infected cells

项目摘要/摘要 B细胞生物学和分子病毒学的进步使这种病毒的发现、鉴定和商业化成为可能 发展广谱中和抗体(BNAbs)。它们是一种很有前途的免疫疗法，可以 纳入了治疗和/或治愈艾滋病毒-1的许多战略。抗体具有多种效应器功能 超越中和和他们治疗/治愈的最重要的作用机制可能是他们的能力 调理感染细胞，标记抗体依赖的细胞毒性或吞噬功能(ADCC/ADCP) 通过免疫效应细胞。目前尚不清楚bNAbs在临床试验中是否在调节感染细胞的清除 它们被被动地注入慢性艾滋病毒感染者(PLWH)。造成这种情况的一个明显原因 BNAbs缺乏药效可能与抑制ART时缺乏Env蛋白表达有关。我们缺少的是 关于HIV潜伏期和潜伏期逆转期间env表达水平的知识阻碍了有效使用 作为治疗学的BNAbs。为了弥补这一差距，在感染细胞中检测低水平环境蛋白表达的能力 需要有不同亚型的艾滋病毒-1。博斯克实验室发布了一种超灵敏的方法来检测p24 GAG蛋白降至Fg/ml水平，并为新开发的Env检测提供了初步数据。在这里，我们建议 在描述良好的潜伏期模型中，使用这些检测方法检测低水平的Env和Gag蛋白，并比较 用流式细胞仪检测Env表面表达的数据(目标1)。在这些化验中，我们有 使用针对不同表位的bNAb来检测环境病毒构象的灵活性(即，三聚体， 单体等)，并将这些分析扩展到来自不同亚型的不同HIV分离株，以衡量差异 可归因于遗传多样性病毒的环境表达和潜伏期(目标2)。了解这些关键因素 关于Env和Gag的表达在仔细控制的潜伏期模型中将使我们能够进一步研究HIV 未来使用PLWH队列进行蛋白质翻译的研究。这些研究将提高治疗学的能力 使用环境靶向策略来靶向潜伏感染的细胞。

Project Summary/Abstract Advances in B cell biology and molecular virology have enabled the discovery, characterization, and commercial development of broadly neutralizing antibodies (bNAbs). They are a promising immunotherapy that can be incorporated in many strategies for the treatment and/or cure of HIV-1. Antibodies have many effector functions beyond neutralization and their most important mechanism of action for treatment/cure may be their ability to opsonize infected cells, tagging them for antibody-dependent cellular cytotoxicity or phagocytosis (ADCC/ADCP) by immune effector cells. It is not clear that bNAbs are mediating clearance of infected cells in clinical trials when they are passively infused into chronically infected people living with HIV (PLWH). An obvious reason for this lack of efficacy of bNAbs may be a lack of Env protein expression while on suppressive ART. Our lack of knowledge about the level of Env expression during HIV latency and latency reversal prevents effective use of bNAbs as therapeutics. To address this gap, the ability to detect low level Env protein expression in cells infected with different subtypes of HIV-1 is needed. The Bosque lab has published an ultrasensitive method to detect p24 Gag protein down to the fg/ml level and has preliminary data for a newly developed Env assay. Here we propose to use these assays to detect low levels of Env and Gag protein in a well-described model of latency and compare the data to Env surface expression as measured by flow cytometry (Aim 1). Within these assays, we have the flexibility to test Env detection using bNAbs targeting different epitopes to probe for Env conformation (i.e., trimer, monomer, etc) and extend these analyses to diverse HIV isolates from different subtypes to measure differences in Env expression and latency attributable to genetically diverse virus (Aim 2). Knowing these crucial factors about Env and Gag expression in a carefully controlled model of latency will allow us to further investigate HIV protein translation in future studies using cohorts of PLWH. These studies will improve the ability of therapeutics using Env-targeting strategies to target latently infected cells.