Biological Properties of HIV-1 V3 Evolutionary Variants

HIV-1 V3 进化变异体的生物学特性

基本信息

批准号：
9321715
负责人：
Ronald I Swanstrom
金额：
$ 39.6万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-01 至 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9321715
关键词：
Address Age Biological Biological Assay Biological Process Biology Blood CD4 Positive T Lymphocytes Cell Count Cells China Clinic Consensus Sequence DNA Data Data Set Defective Viruses Detection Development Dideoxy Chain Termination DNA Sequencing Disease Disease Management Disease Progression Drug resistance Evolution Failure Frequencies Generations Genes Genetic Recombination Genome Genomics Goals Guidelines HIV HIV-1 Hospitals Human Human body Infection Learning Length Link Mediating Menopause Methods Minor Monitor Mutation Natural History Nature Neurons Patients Pattern Plasma Population Population Analysis Population Dynamics Population Heterogeneity Problem Solving Property Residual state Resistance Role Sampling Source Structure Technology Testing Time Treatment Failure Variant Viral Viral Genes Viral Genome Viremia Virion Virus Woman abstracting base biological systems cell type clinical care cohort deep sequencing inflammatory marker insight neutralizing antibody new technology next generation sequencing pathogen quantum resistance mutation screening sequencing platform tool viral RNA whole genome

项目摘要

Project Summary/Abstract Viral gene sequences represent a rich and valuable source of information about biological processes. Advances in next generation sequencing (NGS) technology over the past decade now provide the opportunity to probe viral population diversity in unprecedented ways. We recently developed the Primer ID strategy to overcome several serious limitations of conventional NGS including greatly reducing the mis-incorporation and recombination introduced by the preceding PCR step, reducing the errors of the sequencing platform, and revealing sampling depth of the initial viral genomes/templates that are actually represented in the final data set. In this application we describe how we will use Primer ID with NGS to address long-standing issues in HIV-1 population analysis in the context of viral evolution in the absence of therapy, with failed therapy, and with suppressive therapy. In addition, we will link our sequence data to state-of-the-art evolutionary analysis with a goal of making high quality data sets and tools readily available for further secondary analyses. In Aim 1, we will obtain longitudinal plasma samples of 28 HIV-infected women (from the WIHS cohort) starting with high CD4+ T cell counts until they progress to CD4+ T cell counts of less than 100 cells/µL. We will perform multiplexed Primer ID sequencing to obtain near full length HIV-1 genome. We predict that X4 variants in viral populations first emerge at low abundance that we will be able to detect much earlier than previously observed. In addition, we will investigate longitudinal viral diversity changes in all sequenced regions to assess population dynamics and link all of these markers to markers of inflammation, CNS damage, and the breadth and potency of neutralizing antibodies. In Aim 2, we will apply the multiplexed Primer ID sequencing approach as a screening tool for drug resistance testing. We hypothesize that the potential for drug resistance mutations to mediate escape can occur from minor variants, too minor to be reliably detected with the methods that have been used to date. We will analyze samples from a large cohort of subjects attending the HIV clinic at the #8 Hospital in Guangzhou, China, to determine how often minor variants are missed by assessing therapy failure using Sanger sequencing, determine the role of transmitted or pre-existing mutations in the failure of WHO first line therapy, and define the mutation pattern after subsequent failure of WHO second line therapy. In Aim 3, we will use deep sequencing with Primer ID to monitor population changes in subjects on therapy. We hypothesize that changes in the low level viral populations occur even while on therapy and that these can be monitored using deep. The insights gained will be informative for how to apply this technology to search for evolutionary variants related to X4 viruses, to define other biological correlates of the changing viral population with disease progression in women, to define how the viral population gets established with regard to the steady-state levels of minor sequence variants, to examine the role of drug resistance variants in therapy failure, and to explore the utility of NGS technology in the setting of subjects on therapy.

项目概要/摘要病毒基因序列代表了有关生物过程的丰富且有价值的信息来源。过去十年中下一代测序 (NGS) 技术的进步现在提供了机会我们最近开发了 Primer ID 策略来以前所未有的方式探索病毒种群多样性。克服了传统NGS的几个严重限制，包括大大减少错误掺入和前述PCR步骤引入的重组，减少测序平台的错误，以及揭示最终数据中实际表示的初始病毒基因组/模板的采样深度在此应用程序中，我们描述了如何将 Primer ID 与 NGS 结合使用来解决长期存在的问题。在没有治疗、治疗失败的情况下，在病毒进化的背景下进行 HIV-1 群体分析，以及此外，我们还将把我们的序列数据与最先进的进化分析联系起来。目标是为进一步的二次分析提供高质量的数据集和工具。我们将获得 28 名 HIV 感染女性（来自 WIHS 队列）的纵向血浆样本，从高我们将进行 CD4+ T 细胞计数，直至 CD4+ T 细胞计数低于 100 个细胞/μL。多重引物 ID 测序以获得接近全长的 HIV-1 基因组，我们预测病毒中存在 X4 变异。种群首先以低丰度出现，我们将能够比以前观察到的更早地发现这一点。此外，我们将调查所有测序区域的纵向病毒多样性变化，以评估群体动态并将所有这些标记物与炎症标记物、中枢神经系统损伤以及广度和效力联系起来在目标 2 中，我们将应用多重引物 ID 测序方法作为方法。我们发现耐药突变的潜力。微小的变异可能会发生介导逃逸，这些变异太小而无法用现有的方法可靠地检测到。迄今为止，我们将分析来自在 #8 的 HIV 诊所就诊的大量受试者的样本。中国广州的一家医院，旨在确定通过评估治疗失败而漏掉微小变异的频率使用桑格测序，首先确定传播的或预先存在的突变在世卫组织失败中的作用一线治疗，并确定 WHO 二线治疗随后失败后的突变模式在目标 3 中，我们将使用带有 Primer ID 的深度测序来监测接受治疗的受试者的群体变化。保持低水平病毒种群的变化即使在治疗期间也会发生，并且这些变化可以是使用深度监控获得的见解将为如何应用该技术进行搜索提供信息。与 X4 病毒相关的进化变异，以定义不断变化的病毒群体的其他生物相关性随着女性疾病的进展，确定病毒种群如何在以下方面建立：微小序列变异的稳态水平，以检查耐药变异在治疗中的作用失败，并探索 NGS 技术在治疗受试者设置中的效用。