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

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

• 批准号: 10320584
• 负责人: Melissa Laird Smith
• 金额: $ 17.41万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 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 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10320584
• 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.

项目摘要 中枢神经系统的HIV感染是慢性疾病阶段中发病率和死亡率的重要来源， 尽管有抗逆转录病毒疗法的组合可用性（CART）。尽管早期和广泛使用购物车 减少了更严重的CNS病理的发生，记录了米勒神经认知障碍 在20-50％的感染者中。据认为，艾滋病毒入侵中枢神经系统发生在 感染，病毒感染周围巨噬细胞和小胶质细胞，除了CD4+中央记忆 T细胞（TCCM），是周围感染的主要感染细胞。绝大多数的外围护理人都存在 静止的CD4+ TCCM的一小部分，被认为是通过延伸的克隆膨胀来维持的 感染细胞。远处的艾滋病毒储层表征着重于识别和分类 集成位点或检查综合提供基因组的完整性，很少同时同时同时。潜伏期 研究通常是通过患者来源的PBMC进行的，并且可能无法充分代表独特 组织特异性储层的特征。特别是，对中枢神经系统特异性HIV储藏的研究很少且有限 通过示例访问。脑HIV的扩展细胞向向潮流和低水平，持续病毒的潜力 复制表明，与 等离子体;但是，CNS储层在病毒基因组完整性方面的特征仍然很差， 病毒变体的集成位点，出现和储层固定以及克隆扩张对 储层维护。此外，CD4+ T记忆细胞可以增殖。终末分化的髓样 和神经胶质细胞是大脑中艾滋病毒感染的主要靶标，长期存在，自我的能力有限 更新。用于分析艾滋病毒水库的标准方法取决于简短阅读的下一代 测序（NGS）技术需要检查积分位点必须分化 根据其相关临时基因组的表征。简短读取NGS集成审讯 站点限制了所得数据的可映射性，这可能会进一步限制HIV积分位点的识别； 虽然将单个基因组扩增（SGA）用于临时基因组表征是低吞吐量，而 劳动密集型。在这里，我们开发了一种新颖的创新HIV特异性分子富集方法， 结合单分子测序（“ HIV SMRTCAP”），该测序分辨出完整的HIV“ Integron” （侧翼整合位点和相关的病毒）无论基因组环境如何。我们建议应用此 由国家提供的技术来表征HIV感染的中枢神经系统和淋巴组织 神经辅助组织联盟，包括病毒（n = 3）和抑制CART（n = 3）患者， 首次定义克隆扩张对CNS限制储层的贡献。结果 由拟议项目生成的将建立一种直接询问艾滋病毒整合物的新方法 将允许围绕组织限制的HIV持久性和储层维持的假设产生。