HIV eradication by ADCC-activated NK cell killing

描述（由申请方提供）：为了实现HIV感染的功能性治愈，必须充分降低潜伏病毒储库的大小，以允许不确定地停止抗病毒药物治疗。我们的策略结合了多种已成功测试过的抗癌症和HIV-1的方法，目标是在HAART治疗受到良好抑制的患者中显著减少HIV储库。具体而言，我们将联合收割机体外细胞因子激活自然杀伤（NK）细胞与刺激抗体依赖性细胞介导的细胞毒性（ADCC）使用广泛中和抗体抗HIV-1 Env。Env表达将通过用潜伏期逆转药物诱导前病毒转录来实现，例如目前正在进行临床评价的HDAC抑制剂。 在本申请的R21阶段，我们将使用新开发的HIV潜伏期离体模型来优化策略。在具体目标1中，我们将定义和优化以下方面的特异性： NK细胞介导的对不同CD 4 + T细胞亚群的杀伤。这将涉及使用不同的潜伏期逆转药物测量在CD 4 + T细胞亚群上表达的NK细胞活化/抑制受体配体，测量自体NK细胞杀死潜伏感染的CD 4 + T细胞亚群的功效，以及优化NK细胞的离体细胞因子活化。在具体目标2中，我们将研究使用针对Env的广泛中和抗体刺激ADCC在NK细胞介导的对潜伏性HIV-1感染的原代T细胞的杀伤中与前病毒再激活相结合的有效性。 在完成该项目的R21阶段后，我们将建立可靠的测定方法来测量NK介导的对再活化的潜伏感染T细胞的杀伤，定义体外活化NK细胞的方案，并鉴定用于ADCC介导的杀伤的抗体。本申请的R33阶段将专门关注使用患者细胞的离体研究，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HIV诊所，特殊免疫科（SIU，UHCMC）招募。SIU有1 026名艾滋病毒感染者，他们定期接受常规医疗护理;大约90%的人接受抗逆转录病毒治疗。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来自HIV感染者的自体原代T细胞的杀伤。在具体目标4中，我们将评估目标1、2和3中开发的技术是否也导致HIV感染患者中潜伏内源性病毒库的减少。我们将在存在和不存在ADCC抗体、存在和不存在T细胞诱导以及存在和不存在NK细胞活化的情况下对NK杀伤进行成对比较。将分析至少10份患者样本，以确保结果具有统计学显著性。检测试剂盒的读数将是通过下一代测序分析的定量PCR反应的定量HIV前病毒DNA和mRNA测量值。 该提案中的实验将为NK导向根除潜伏HIV的临床研究奠定基础。由于我们的策略结合了使用可用抗体通过ADCC增强NK选择性、NK细胞的离体活化和潜伏前病毒的体内活化，我们认为它代表了一种实用的解决方案，可以在临床上容易地对大量患者实施。

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.