Characterization of clonal expansion in the CNS-restricted reservoir using HIV SMRTcap, a novel single molecule assay providing simultaneous resolution of proviral genomes and integration sites

项目总结 中枢神经系统的艾滋病毒感染是慢性病阶段发病率和死亡率的重要来源， 尽管有联合抗逆转录病毒疗法(CART)可用。尽管购物车的早期和广泛使用 减少更严重的中枢神经系统病理的发生，记录较轻的神经认知障碍 在20%-50%的感染者中。艾滋病毒对中枢神经系统的入侵被认为发生在 感染，除CD4+中枢记忆外，病毒还感染血管周围巨噬细胞和小胶质细胞 T细胞(TCCM)，外周主要感染细胞。绝大多数外围储集层以 静息的CD4+TCCM中非常小的一部分，被认为是通过潜伏的克隆性扩张而维持的 被感染的细胞。到目前为止，艾滋病毒携带者的特征描述侧重于识别和分类 整合位点或检查整合的前病毒基因组的完整性，很少同时进行。潜伏期 大多数研究是用患者来源的PBMC进行的，可能不能充分代表独特的 特定组织储集层的特征。特别是，对中枢神经系统特有的艾滋病毒储备库的研究很少，而且有限 通过样本访问。脑部HIV的细胞扩张性和低水平持续病毒的可能性 重复表明，与北半球相比，中南半球的储集层动力学可能有很大的不同 血浆；然而，中枢神经系统储存库在前病毒基因组完整性方面的特征仍然很差， 病毒变异的整合部位、出现和储存固定以及克隆扩张对 水库维护。此外，虽然CD4+T记忆细胞可以增殖；但终末分化的髓样细胞 而脑中HIV感染的主要目标神经胶质细胞寿命长，自我调节能力有限。 更新。描述HIV宿主的标准方法依赖于下一代短读数 需要检查整合位点的测序(NGS)技术必须分离 根据它们相关的前病毒基因组的特征。短文阅读NGS集成审问 地点限制了结果数据的可测绘能力，这可能进一步限制艾滋病毒整合地点的确定； 虽然使用单基因组扩增(SGA)来描述前病毒基因组特征是低吞吐量和 劳动密集型。在这里，我们开发了一种新颖的、创新的HIV特异性分子浓缩方法， 结合单分子测序(“HIV SMRTcapp”)，它解决了完整的HIV“整合子” (侧翼整合位点和相关的前病毒)，与基因组背景无关。我们建议将这一点应用于 HIV感染中枢神经系统和淋巴组织的鉴定技术，由国家 神经艾滋病组织联盟，包括病毒携带者(n=3)和CART抑制者(n=3)， 首次确定克隆扩张对中枢神经系统受限储层的贡献。结果是 将建立一种新的方法来直接询问HIV整合子和 将允许围绕组织限制性艾滋病毒持久性和水库维护产生假说。

PROJECT SUMMARY HIV infection of the CNS is an important source of morbidity and mortality in the chronic phase of disease, despite availability of combination Antiretroviral Therapy (cART). Although early and wide use of cART has reduced the occurrence of the more severe CNS pathologies, milder neurocognitive impairment is documented in 20-50% of infected individuals. HIV invasion of the CNS is thought to occur within the first two weeks of infection, where the virus infects perivascular macrophages and microglia, in addition to CD4+ central memory T cells (TCCM), the primary infected cell in the periphery. The vast majority of the peripheral reservoir exists as a very minor fraction of resting CD4+ TCCM and is thought to be maintained by the clonal expansion of latently infected cells. Thus far, HIV reservoir characterization has focused on either identifying and classifying integration sites or examining the integrity of the integrated proviral genomes, rarely both concurrently. Latency studies are most often performed with patient-derived PBMC and may not adequately represent unique features of tissue-specific reservoirs. In particular, studies of the CNS-specific HIV reservoir are few and limited by sample access. The expanded cell tropism of brain HIV and the potential for low level, ongoing viral replication are suggestive that reservoir dynamics may be dramatically different in the CNS compared to plasma; however, the CNS reservoir remains poorly characterized with regard to proviral genome integrity, integration sites, emergence and reservoir fixation of viral variants and the contribution of clonal expansion to reservoir maintenance. Moreover, while CD4+ T memory cells can proliferate; terminally differentiated myeloid and glial cells, the primary targets of HIV infection in the brain, are long-lived with limited capacity for self- renewal. Standard methods for profiling HIV reservoirs are dependent on short read next generation sequencing (NGS) technologies that require the examination of integration sites to be necessarily divorced from the characterization of their associated proviral genomes. Short read NGS interrogation of integration sites limits the mapability of the resulting data, which may further limit the identification of HIV integration sites; while the use of single genome amplification (SGA) for proviral genome characterization is low throughput and labor intensive. Here we have developed a novel, innovative HIV-specific molecular enrichment approach, combined with single molecule sequencing (“HIV SMRTcap”), which resolves the complete HIV “integron” (flanking integration sites and associated provirus) regardless of genomic context. We propose to apply this technology to the characterization of HIV-infected CNS and lymphoid tissues, provided by the National NeuroAIDS Tissue Consortium and including both viremic (n=3) and cART suppressed (n=3) patients, to define, for the first time, the contribution of clonal expansion to the CNS-restricted reservoir. The results generated by the proposed project will establish a novel method for directly interrogating the HIV integron and will permit hypothesis generation surrounding tissue-restricted HIV persistence and reservoir maintenance.