Ultrasensitive Single-Genome Sequencing to Study HIV Transmission and Evolution

背景：在本项目中，我们开发了一种新的病毒基因组NGS文库构建方法，该方法减少了PCR偏置和错误，从最终数据集中消除了PCR重组，并生成了数千个与标准测序质量相同但变体多100倍的单基因组HIV序列（Boltz et al., Retrovirology 13:87, 2016）。这种新的、超灵敏的SGS （uSGS）检测不仅用于检测罕见等位基因的连锁，包括耐药突变（Boltz等）。JCI Insight 2021)，也可用于病毒种群的深入系统发育分析。尽管人们普遍认为大多数HIV感染是由单个病毒粒子引发的，但这一结论是基于分析的病毒基因组数量相对较少（约10-100个HIV基因组）的实验得出的。我们将利用美国地质调查局的方法对急性感染中的HIV遗传学进行深入研究。潜在的问题是，在一些似乎感染了单一病毒的个体中，是否也存在由于采样浅而遗漏的少数变异。我们能够从参加RV254/东南亚研究合作与夏威夷010试验的15名献血者那里获得血浆样本（101）。这项试验是由泰国红十字会艾滋病研究中心和美国陆军医疗部门、武装部队医学科学研究所逆转录病毒部进行的。该研究旨在确定泰国高危人群中的急性艾滋病毒感染，并提供立即开始抗逆转录病毒治疗。从急性HIV感染期间的参与者中收集了15个样本。以前报告的10名捐献者在标准SGS急性感染中只有一种创始人病毒，而使用相同方法的5名捐献者显示有两到五个创始人病毒。在我们的研究中，我们将调查急性HIV感染中单个创始人病毒的存在与多个创始人病毒的传播。总的来说，我们的目标是从每个供体样本中获得10,000个HIV基因组，从而对急性HIV感染的遗传学进行最深入的研究。这一目标的结果将为使用bNAbs在急性感染中靶向创始病毒的研究提供信息，并将有助于我们了解HIV的传播、进化和种群动态。除了我们将uSGS应用于传播和进化研究的目标之外，我们还将使用牛津纳米孔技术（ONT）开发下一代uSGS。我们已经开始了这些研究，证明ONT可以用于生成准确的接近全长的HIV测序（Wright等）。细胞2021)，并通过开发一个管道来注释生成的序列（Wright等人）。Retrovirology 2021)。展望未来，我们将改进目前的ONT检测方法和管道，以捕获艾滋病毒整合位点以及从接受抗逆转录病毒治疗的供体收集的样本中的全长艾滋病毒序列。这一进展将大大有助于我们了解艾滋病毒的持久性。Freed博士最近报告说，HIV可以通过获得Env突变，在体外对多种抗逆转录病毒药物产生耐药性。然而，目前尚不清楚这些突变是否在体内像在体外一样赋予多药耐药性。为了研究Env突变对体内耐药的可能贡献，我们获得了5名供者的血浆样本，这些供者未能通过标准HIV基因分型获得含有雷替格拉韦（raltegravir）的INSTI（整合酶基因无突变）方案，且血浆药物水平证实其依从性。使用ACTG研究A5273获得的样本，我们目前正在对art前、早期RAL衰竭和晚期RAL衰竭的供体样本进行uSGS。迄今为止获得的序列分析了已知的in耐药突变和新出现的Env突变。高度保守的gp120/gp41界面在药物失败后出现了几个Env突变，类似于那些在体外被证实赋予耐药性的突变（Hikichi, et al. mBio 2021）。这些初步发现表明，Env突变可能有助于体内HIV的耐药性。如果Env突变被证明对抗逆转录病毒疗法产生耐药性，我们将启动进化研究，以确定新发现的耐药性突变的起源和选择压力（例如，它们是否在抗逆转录病毒疗法之前存在，如果存在，频率是多少，它们是否在未能接受其他抗逆转录病毒疗法的供者中发现，以及它们是否在未接受抗逆转录病毒疗法的供者中被选中）。了解Env在抗逆转录病毒治疗耐药中的作用将具有重要的临床意义，如果发现突变单独产生耐药性或成为病毒学失败的“垫脚石”。____成就：通过我们的SGS检测，我们证明，当病毒变体的供体人群中存在耐药突变时，暴露前预防可能无法预防HIV传播（Spinelli等人，临床）。感染。Dis. 72:2025- 2028,2021)。我们与Eric Freed博士（HIV Dynamics and Replication Program）的实验室合作，研究了脱靶突变在ART无法控制HIV在体内复制中的作用。我们在HIV env基因的gp120-gp41界面上发现了几个突变，这些突变能够赋予对多种抗逆转录病毒药物低至中等水平的耐药性（Hikichi等人，mBio12:e03134- 20,2021）。我们使用Oxford Nanopore Technologies开发了一种新的近全长HIV单基因组测序方法，并证明其准确性与其他方法相当，成本更低，通量更高。该试验将用于表征抗逆转录病毒治疗中持续存在的HIV原病毒（Wright等）。细胞。10,2021)。我们开发了一种新的生物信息学管道来注释接近全长的HIV序列，并确定它们是否完整，是否有可能在ART中断时加速反弹病毒血症（Wright等）。逆转录病毒学[j] .浙江大学学报（自然科学版）。

BACKGROUND: In this project, we developed a new method for NGS library construction of viral genomes that reduces PCR bias and error, eliminates PCR recombinants from the final datasets, and generates thousands of single-genome HIV sequences of the same quality as standard sequencing but with 100-fold more variants (Boltz et al., Retrovirology 13:87, 2016). This new, ultrasensitive SGS (uSGS) assay has been used not only for detecting linkage of rare alleles, including drug-resistance mutations (Boltz, et al. JCI Insight 2021), but also for in-depth phylogenetic analyses of viral populations. Although it is generally believed that most HIV infections are initiated with a single virion, this conclusion rests on experiments in which the number of viral genomes analyzed was relatively small (about 10-100 HIV genomes). We will utilize our uSGS method to perform a deep-dive study of HIV genetics in acute infection. The underlying question is whether, in some of the individuals that appear to have been infected with a single virus, there were also minority variants that were missed due to shallow sampling. We were able to obtain plasma samples from 15 donors enrolled in the RV254/South East Asia Research Collaboration with Hawaii 010 Trial (101). The trial was conducted by the Thai Red Cross AIDS Research Centre and the Department of Retrovirology, U.S. Army Medical Component, Armed Forces Research Institute of Medical Sciences. The study was designed to identify acute HIV infection in high-risk populations in Thailand and offer immediate initiation of ART. Fifteen samples were collected from participants during acute HIV infection. Ten of the donors were previously reported to have a single founder virus in acute infection by standard SGS, and five donors were shown to have two to five founders using the same approach. In our study, we will investigate the presence of a single founder virus verses transmission of multiple founders in acute HIV infection. In total, we aim to obtain 10,000 HIV genomes from each donor sample, constituting the deepest look into the genetics of acute HIV infection. Results from this aim will inform studies using bNAbs in acute infection to target founder viruses and will contribute to our understanding of HIV transmission, evolution, and population dynamics. In addition to our goals of applying uSGS to studies of transmission and evolution, we will also develop the next generation of uSGS using Oxford Nanopore Technologies (ONT). We have begun these studies by demonstrating that ONT can be used to generate accurate near full-length HIV sequencing (Wright, et al. Cells 2021) and by developing a pipeline to annotate the sequences generated (Wright, et al. Retrovirology 2021). Moving forward, we will improve our current ONT assays and pipeline to capture the HIV integration sites as well as the full-length HIV sequences in samples collected from donors on ART. This advancement will contribute significantly to our understanding of HIV persistence. Dr. Freed recently reported that HIV can develop drug resistance in vitro to multiple classes of antiretroviral drugs by acquiring Env mutations. However, it is not known if these mutations confer multidrug resistance in vivo as they do in vitro. To investigate the possible contribution of Env mutations to drug resistance in vivo, we obtained plasma samples from five donors failing INSTI [raltegravir (RAL)]-containing regimens without mutations in the integrase gene by standard HIV genotyping and with plasma drug levels confirming adherence. Using samples obtained from the ACTG study A5273, we are currently performing uSGS on donor samples from pre-ART, early RAL failure, and during late RAL failure. Sequences obtained thus far were analyzed for known drug resistance mutations in IN and for newly emergent Env mutations. Several Env mutations emerged upon drug failure in the highly conserved gp120/gp41 interface, similar to those confirmed to confer drug resistance in vitro (Hikichi, et al. mBio 2021). These preliminary findings suggest that Env mutations might contribute to HIV drug resistance in vivo. If Env mutations are demonstrated to confer resistance to ART, we will initiate evolutionary studies to determine the origin and selection pressures on the newly identified drug resistance mutations (e.g., if they existed prior to ART and, if so, at what frequencies, if they are found in donors failing other ART regimens, and if they are selected in donors without ART). Understanding the role of Env in ART resistance will have significant clinical impact if mutations are found to confer drug resistance alone or to be a "stepping stone" toward virologic failure. ____ACCOMPLISHMENTS: ___Using our SGS assays, we demonstrated that pre-exposure prophylaxis can fail to prevent HIV transmission when drug-resistance mutations are present in the donor population of viral variants (Spinelli et al., Clin. Infect. Dis. 72:2025-2028, 2021). ___In collaboration with the lab of Dr. Eric Freed (HIV Dynamics and Replication Program), we investigated the role of off-target mutations in the failure of ART to control HIV replication in vivo. We identified several mutations in the gp120-gp41 interface of the HIV env gene that are capable of conferring low- to medium-level resistance to multiple ARVs (Hikichi et al., mBio12:e03134-20, 2021). ___We developed new near full-length HIV single-genome sequencing assay using Oxford Nanopore Technologies and demonstrated it's accuracy to be equivalent to other approaches at a reduce cost and higher throughput. This assay will be used to characterize HIV proviruses that persist on ART (Wright, et al. Cells. 10, 2021). ___We developed a new bioinformatic pipeline to annotate near full-length HIV sequences and determine if they are intact and likely capable of fueling rebound viremia if ART is interrupted (Wright, et al. Retrovirology. 18: 16, 2021.).