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

使用 HIV SMRTcap 表征 CNS 限制的 HIV 储存库中的克隆扩增，这是一种新型单分子检测，可同时解析原病毒基因组和整合位点

• 批准号: 9927047
• 负责人: Melissa Laird Smith
• 金额: $ 25.43万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-06 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9927047
• 关键词: AIDS/HIV problem; Autopsy; Biological Assay; Blood; Brain; CD4 Positive T Lymphocytes; Cell Culture Techniques; Cell Line; Cells; Central Nervous System Infections; Chronic; Chronic Phase of Disease; Clonal Expansion; Custom; Data; Disease; Divorce; Event; Evolution; Frequencies; Generations; Genes; Genetic Transcription; Genome; Genomic DNA; Genomics; HIV; HIV Infections; HIV-1; Immune; Individual; Infection; Integrons; Introns; Knowledge; Length; Lymphoid; Lymphoid Tissue; Maintenance; Maps; Mediating; Methodology; Methods; Microglia; Minor; Modeling; Molecular; Morbidity - disease rate; Myeloid Cells; National NeuroAids Tissue Consortium; Nature; Neuraxis; Neurocognitive Deficit; Neuroglia; Pathology; Patients; Performance; Peripheral; Peripheral Blood Mononuclear Cell; Plasma; Play; Population; Proliferating; Proviruses; Reproducibility; Resolution; Rest; Role; Sampling; Sensitivity and Specificity; Source; Suggestion; T memory cell; Technology; Testing; Time; Tissue Sample; Tissues; Tropism; Variant; Viral; Virus; Virus Replication; analysis pipeline; antiretroviral therapy; cell type; defined contribution; experimental study; genome integrity; innovation; integration site; macrophage; member; memory CD4 T lymphocyte; mortality; next generation sequencing; novel; novel strategies; sample fixation; self-renewal; single molecule; single molecule real time sequencing; site-specific integration

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.

项目概要 中枢神经系统的艾滋病毒感染是疾病慢性期发病率和死亡率的重要来源， 尽管可以使用联合抗逆转录病毒疗法 (cART) 尽管 cART 已得到早期和广泛的使用。 减少了更严重的中枢神经系统病变的发生，记录了更轻微的神经认知障碍 20-50% 的感染者中，HIV 入侵中枢神经系统被认为发生在感染后的前两周内。 感染，除了 CD4+ 中央记忆外，病毒还感染血管周围巨噬细胞和小胶质细胞 T细胞（TCCM），外周的主要感染细胞 绝大多数外周储存库存在。 静息 CD4+ TCCM 的一小部分，并且被认为是通过潜伏的克隆扩增来维持的 到目前为止，HIV 储存库特征主要集中在识别和分类上。 整合位点或检查整合原病毒基因组的完整性，很少同时进行。 研究最常使用患者来源的 PBMC 进行，可能不足以代表独特的情况 特别是，对 CNS 特异性 HIV 病毒库的研究很少且有限。 通过样本获取，脑部艾滋病毒的细胞向性扩大以及低水平、持续病毒的潜力。 复制表明，与中枢神经系统相比，储层动力学可能显着不同 血浆；然而，中枢神经系统储存库的前病毒基因组完整性仍不清楚， 病毒变体的整合位点、出现和储存库固定以及克隆扩增的贡献 此外，CD4+ T记忆细胞可以增殖终末分化的骨髓； 神经胶质细胞是大脑中 HIV 感染的主要目标，它们寿命较长，但自我修复能力有限。 HIV病毒储存库分析的标准方法依赖于下一代短读长。 要求检查整合位点必须分离的测序 (NGS) 技术 来自其相关原病毒基因组的短读 NGS 整合分析。 位点限制了结果数据的可映射性，这可能进一步限制艾滋病毒整合位点的识别； 而使用单基因组扩增 (SGA) 进行原病毒基因组表征的通量较低且 在这里，我们开发了一种新颖的、创新的艾滋病毒特异性分子富集方法， 结合单分子测序（“HIV SMRTcap”），解析完整的HIV“整合子” （侧翼整合位点和相关原病毒）无论基因组背景如何，我们都建议应用这一点。 技术来表征艾滋病毒感染的中枢神经系统和淋巴组织，由国家提供 NeuroAIDS 组织联盟，包括病毒血症 (n=3) 和 cART 抑制 (n=3) 患者，以 首次定义克隆扩张对 CNS 限制性储存库的贡献。 拟议项目产生的结果将建立一种直接询问艾滋病毒整合子的新方法 将允许围绕组织限制的 HIV 持久性和储存库维护产生假设。