HIV eradication by ADCC-activated NK cell killing

描述(由申请人提供)：为了实现对艾滋病毒感染的功能性治愈，必须充分减少潜伏的病毒库的大小，以允许无限期地停止抗病毒药物治疗。我们的战略结合了多种已经成功测试抗癌和HIV-1的方法，目标是显著减少HAART抑制良好的患者中的HIV蓄积。具体地说，我们将使用针对HIV-1Env的广谱中和抗体，将自然杀伤(NK)细胞的体外细胞因子激活与抗体依赖细胞介导的细胞毒(ADCC)刺激相结合。Env的表达将通过潜伏期逆转药物诱导前病毒转录实现，例如目前正在进行临床评估的HDAC抑制剂。在这项应用的R21阶段，我们将使用新开发的艾滋病毒潜伏期体外模型来优化策略。在具体目标1中，我们将定义和优化 NK细胞对不同CD4+T细胞亚群的杀伤作用这将涉及使用不同的潜伏期逆转药物测量表达在CD4+T细胞亚群上的NK细胞激活/抑制受体配体，测量自体NK细胞杀死潜伏感染的CD4+T细胞亚群的效率，以及优化NK细胞的体外细胞因子激活。在特定的目标2中，我们将研究在NK细胞介导的对潜伏感染HIV-1的初级T细胞的杀伤中使用针对Env的广谱中和抗体来刺激ADCC的有效性，并结合前病毒的重新激活。在这个项目的R21阶段完成后，我们将建立可靠的检测方法来测量NK介导的对再次激活的潜伏感染T细胞的杀伤，定义体外激活NK细胞的方案，并识别用于ADCC介导的杀伤的抗体。这项应用的R33阶段将专门专注于使用患者细胞进行的体外研究，这些细胞旨在显示潜伏感染细胞的消除。人类受试者将从我们的艾滋病毒诊所，特别免疫科(SIU，UHCMC)招募。SIU有1026名艾滋病毒感染患者的活跃人口，他们定期接受常规医疗护理；大约90%正在接受抗逆转录病毒治疗(ART)。在具体目标3中，我们将验证R21阶段的技术是否可以增强对艾滋病毒感染者的自体初级T细胞的杀伤。在具体的目标4中，我们将评估在目标1、2和3中开发的技术是否也导致艾滋病毒感染患者的潜在内源性病毒库的减少。我们将对存在和不存在ADCC抗体、有和没有T细胞诱导以及有和没有NK细胞激活情况下的NK杀伤进行成对比较。将对至少10个患者样本进行分析，以确保结果具有统计学意义。这些检测的读数将是由下一代测序分析的定量聚合酶链式反应的艾滋病毒前病毒DNA和mRNA的定量测量结果。这项提议中的实验将为NK指导根除潜伏的艾滋病毒的临床研究奠定基础。由于我们的策略结合了利用现有抗体通过ADCC提高NK选择性，体外激活NK细胞和体内激活潜伏前病毒，我们相信它代表了一种实用的解决方案，可以很容易地在临床上对大量患者实施。

DESCRIPTION (provided by applicant): In order to achieve a functional cure for HIV infections the size of the latent viral reservoir must be sufficiently reduced to allow an indefinie cessation of antiviral drug treatment. Our strategy combines a variety of approaches that have been successfully tested against cancer and HIV-1 with the goal of achieving a significant reduction of HIV reservoirs in patients who are well suppressed on HAART. Specifically, we will combine ex vivo cytokine activation of natural killer (NK) cells with stimulation of antibody-dependent cell-mediated cytotoxicity (ADCC) using broadly neutralizing antibodies against HIV-1 Env. Env expression will be achieved by induction of proviral transcription with latency reversal drugs, such as HDAC inhibitors that are currently undergoing clinical evaluation. During the R21 phase of this application we will use newly developed ex vivo models of HIV latency to optimize the strategy. In Specific Aim 1, we will define and optimize the specificity of NK cell-mediated killing of different CD4+ T cell subsets. This will involve measuring NK cell-activating/-inhibiting receptor ligands expressed on CD4+ T cell subsets using different latency-reversing drugs, measuring the efficacy of autologous NK cells to kill latently infected CD4+ T cell subsets, and optimizing ex vivo cytokine activation of NK cells. In Specific Aim 2, we will investigate the effectiveness of using broadly neutralizing antibodies against Env to stimulate ADCC in NK cell-mediated killing of latently HIV-1-infected primary T cells, in combination with proviral reactivation. Upon completion of the R21 phase of this project we will have established reliable assays to measure NK-mediated killing of reactivated latently infected T cells, defined protocols for activating NK cells ex vivo, and identified antibodies for use in ADCC-mediated killing. The R33 phase of this application will focus exclusively on ex vivo studies using patient cells that are designed to show elimination of latently infected cells. Human subjects will be recruited from our HIV clinic, the Special Immunology Unit (SIU, UHCMC). The SIU has an active population of 1026 HIV-infected patients who are followed regularly for routine medical care; approximately 90% are receiving antiretroviral therapy (ART). In Specific Aim 3, we will validate whether techniques during the R21 phase can enhance the killing of autologous primary T cells from HIV-infected individuals. In Specific Aim 4, we will assess whether techniques developed in aims 1, 2, and 3 also lead to a reduction in the latent endogenous viral reservoir in HIV-infected patients. We will perform pairwise comparisons of NK-killing in the presence and absence of antibody for ADCC, with and without T-cell induction and with and without NK cell activation. At least 10 patient samples will be analyzed to ensure statistical significance of the results. The readout for the assays will be quantitative HIV proviral DNA and mRNA measurements from quantitative PCR reactions analyzed by next-generation sequencing. The experiments in this proposal will set the stage for clinical studies of NK-directed eradication of latent HIV. Because our strategy combines enhancing NK selectivity by ADCC using available antibodies, ex vivo activation of NK cells and in vivo activation of latent proviruses, we believe it represents a practical solution that can be readily implemented in the clinic for a large number of patients.