Single Cell Characterization of Latent HIV-1 Reservoirs

 描述（由申请人提供）：HIV-1根除的一个主要障碍是尽管进行了抗逆转录病毒治疗（ART），但仍存在潜伏感染细胞。目前的根除策略集中在用各种潜伏再活化剂（LRA）再活化和清除感染的细胞。然而，单独的病毒再活化不足以减少HIV-1 DNA储库，并且对细胞相关HIV-1 RNA增加与再活化细胞频率之间的关联知之甚少。目前还不清楚再激活策略和HIV-1基因组完整性如何影响HIV-1转录的频率或幅度。因此，我们已经开发并实施了一种新的方法，提供了对HIV-1持久性的深入了解，这是现有技术无法实现的。将单个细胞封装到纳升规模的反应液滴中，然后进行液滴内裂解和未剪接（us）和多重剪接（ms）HIV-1 RNA的PCR扩增，以及基因组病毒DNA的下游分离和测序。我们已经成功地应用这种方法来确定转录活性的CD 4 + T细胞从HIV-1感染的患者抑制性ART与LRA刺激。我们的数据表明，感染细胞的频率可能会增加，而总细胞相关水平下降，反之亦然。我们还观察到usRNA和msRNA对潜在储库激活的反应之间的二分法。这些研究结果表明，单细胞分析将是至关重要的，在提供洞察哪些细胞和组织是目标的根除“休克和杀死”的方法。 我们建议利用我们的检测方法对从早期和晚期ART治疗个体获得的潜伏感染外周血和有组织淋巴组织中分离的细胞进行高通量储库定量和下游HIV-1基因组表征。我们假设效应细胞将显示更高的频率和幅度的HIV-1 RNA再激活比那些记忆或调节表型。早期治疗的个体被认为具有较小的储库，优选地在效应记忆细胞中观察到，其可能具有更有效地反应HIV-1的倾向。我们的目标是：（1）确定在来自受抑制患者的外周血和有组织淋巴组织的单细胞中响应于各种HIV-1潜伏期逆转剂的HIV-1 RNA转录活性，（2）确定在早期和晚期抗逆转录病毒治疗的个体中对离体LRA再活化的单细胞应答，和（3）确定单细胞HIV-1基因序列完整性之间的关系，和HIV-1转录活性。在未来的研究中，我们计划应用我们的新方法来表征HDACi和其他免疫调节疗法的干预性试验的体内反应。我们还计划调整我们的检测方法，以直接测量单个细胞中的病毒生长。

 DESCRIPTION (provided by applicant): A major obstacle to HIV-1 eradication is the existence of latently infected cells that persist despite antiretroviral therapy (ART). Current eradication strategies focus on the reactivation and clearance of infected cells with various latency reactivating agents (LRA). However, viral reactivation alone is insufficient to reduce HIV-1 DNA reservoirs, and the association between increased cell-associated HIV-1 RNA and the frequency of reactivated cells is poorly understood. It is also unknown how reactivation strategies and HIV-1 genome integrity affect the frequency or amplitude of HIV-1 transcription. As a result, we have developed and implemented a novel method that provides insights into HIV-1 persistence that are inaccessible though existing technologies. Individual cells are encapsulated into nanoliter-scale reaction droplets, followed by intra-droplet lysis and PCR amplification of unspliced (us) and multiply spliced (ms) HIV-1 RNA and downstream isolation and sequencing of genomic viral DNA. We have successfully applied this method to identify transcriptionally active CD4+ T cells from HIV-1-infected patients on suppressive ART with and without LRA stimulation. Our data suggest the frequency of infected cells may increase while total cell-associated levels decrease and vice versa. We also observed dichotomies between usRNA and msRNA responses to latent reservoir activation. These findings suggest that single-cell analysis will be crucial in providing insight into which cells and tissues are targets for eradication in "shock and kill" approaches. We propose to utilize our assay to perform high-throughput reservoir quantification and downstream HIV-1 genome characterization of cells isolated from latently-infected peripheral blood and organized lymphoid tissue obtained from early and late ART treated individuals. We hypothesize that effector cells will display higher frequencies and amplitudes of HIV-1 RNA reactivation than those with memory or regulatory phenotype. Early treated individuals are thought to have smaller reservoirs, observed preferentially in effector memory cells which may have the propensity to reactive HIV-1 more efficiently. Our aims are to: (1) determine HIV-1 RNA transcriptional activity in response to various HIV-1 latency reversing agents in single cells derived from peripheral blood and organized lymphoid tissues from suppressed patients, (2) determine the single-cell responses to ex vivo LRA reactivation in early and late antiretroviral treated individuals, and (3) define the relationship between single-cell HIV-1 genetic sequence integrity, and HIV-1 transcriptional activity. In future studies, we plan to apply our novel methods to characterize the in vivo responses to interventional trials of HDACi and other immune modulating therapies. We also plan to adapt our assay to directly measure viral outgrowth in individual cells.