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

项目摘要 CNS的HIV感染是疾病慢性期发病率和死亡率的重要来源， 尽管联合抗逆转录病毒疗法（cART）的可用性。尽管早期和广泛使用cART， 降低了更严重的CNS病理的发生率，记录了较轻的神经认知障碍 20-50%的感染者。HIV对中枢神经系统的侵袭被认为发生在感染后的前两周内。 感染，其中病毒感染血管周围巨噬细胞和小胶质细胞，以及CD 4+中央记忆 T细胞（TCCM），外周中的主要感染细胞。绝大多数外围储层以 静息CD 4 + TCCM的一个非常小的部分，被认为是通过潜伏性的克隆扩增来维持的。 被感染的细胞到目前为止，艾滋病病毒库的特征主要集中在识别和分类 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。延迟 研究最常使用患者来源的PBMC进行， 组织特异性储库的特征。特别是，对中枢神经系统特异性HIV储库的研究很少且有限 样品访问脑HIV的扩展细胞嗜性和低水平、持续病毒感染的可能性 复制表明，与对照组相比，CNS中的储层动力学可能显著不同。 血浆;然而，CNS储库在前病毒基因组完整性方面仍然表征不佳， 整合位点、病毒变异体的出现和储存库固定以及克隆扩增对 水库维护此外，虽然CD 4 + T记忆细胞可以增殖，但终末分化的髓样细胞可以增殖。 神经胶质细胞是艾滋病毒感染的主要目标，它们的寿命很长，自我免疫的能力有限， 退款用于分析HIV储库的标准方法依赖于短读下一代 测序（NGS）技术，需要检查整合位点，必须分离 从其相关的前病毒基因组的特征。集成的短读NGS询问 位点限制了所得数据的可映射性，这可能进一步限制了对艾滋病毒整合位点的识别; 而使用单基因组扩增（SGA）进行前病毒基因组表征是低通量的， 劳动密集型。在这里，我们开发了一种新颖的、创新的HIV特异性分子富集方法， 结合单分子测序（“HIV SMRTcap”），其解析完整的HIV“整合子” （侧翼整合位点和相关的原病毒），而不管基因组背景。我们建议将此应用于 技术的艾滋病毒感染的中枢神经系统和淋巴组织的特征，由国家提供的 NeuroAIDS组织联盟，包括病毒血症（n=3）和cART抑制（n=3）患者， 首次定义了克隆扩张对CNS限制性水库的贡献。结果 该项目将建立一种新的方法来直接询问HIV整合子， 将允许围绕组织限制的HIV持久性和水库维护的假设生成。

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.