Ultrasensitive Single-Genome Sequencing to Study HIV Transmission and Evolution

背景技术背景：在该项目中，我们开发了一种用于病毒基因组的NGS文库构建的新方法，该方法减少了PCR偏倚和错误，从最终数据集中消除了PCR重组体，并产生了数千个与标准测序质量相同但具有100倍以上变体的单基因组HIV序列（Boltz et al.，Retrovirology 13：87，2016）。这种新的超灵敏SGS（uSGS）检测方法不仅用于检测罕见等位基因的连锁，包括耐药突变（Boltz等人，JCI Insight 2021），还用于病毒群体的深入系统发育分析。我们还在使用Oxford Nanopore Technologies（ONT）开发下一代uSGS。我们已经通过证明ONT可用于生成准确的近全长HIV测序（Wright等人，Cells 2021）并通过开发一个管道来注释生成的序列（Wright等人，Retrovirology 2021）开始了这些研究。展望未来，我们将改进我们目前的ONT检测方法和管道，以捕获HIV整合位点以及从ART供体收集的样本中的全长HIV序列。这一进步将大大有助于我们对HIV持久性的理解。Freed博士最近报告说，艾滋病毒可以通过获得Env突变，在体外对多种抗逆转录病毒药物产生耐药性。然而，尚不清楚这些突变是否在体内赋予多药耐药性，因为它们在体外。为了研究Env突变对体内耐药性的可能贡献，我们从5名未通过标准HIV基因分型且血浆药物水平证实依从性的含RHI [雷特格韦（RAL）]方案的供体中获得了血浆样本，其中整合酶基因无突变。使用从ACTG研究A5273中获得的样本，我们对ART前、早期RAL失败和晚期RAL失败期间的供体样本进行测序。对获得的序列分析IN中已知的耐药突变和新出现的Env突变。药物失效后，在高度保守的gp 120/gp 41界面出现了几种Env突变，与体外证实产生耐药性的突变相似（Hikichi等人，mBio 2021）。这些发现表明Env突变可能有助于体内HIV耐药性。如果Env突变被证明赋予对ART的抗性，我们将启动进化研究，以确定新鉴定的耐药突变的起源和选择压力（例如，它们是否在ART之前就存在，如果存在，频率如何，它们是否在其他ART方案失败的供体中发现，以及它们是否在没有ART的供体中被选择）。了解Env在ART耐药性中的作用将具有重要的临床影响，如果发现突变单独赋予耐药性或成为病毒学失败的“垫脚石”。_完成：_使用我们的SGS检测，我们证明了当病毒变体的供体群体中存在耐药性突变时，暴露前预防可能无法预防HIV传播（Spinelli et al.，临床感染72：2025-2028，2021）。_与Eric Freed博士（HIV动力学和复制计划）的实验室合作，我们研究了脱靶突变在ART未能控制体内HIV复制中的作用。我们鉴定了HIV env基因的gp 120-gp 41界面中的几个突变，其能够赋予对多种ARV的低至中等水平抗性（Hikichi等人，mBio12：e03134-20，2021）。_我们使用Oxford Nanopore Technologies开发了新的近全长HIV单基因组测序检测，并证明其准确性与其他方法相当，成本更低，通量更高。该测定将用于表征在ART上持续存在的HIV前病毒（Wright等人，Cells. 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，以及如果ART中断，它们是否有可能引发反弹病毒血症（Wright等人，Retrovirology. 18：16，2021.）。尽管人们普遍认为大多数艾滋病毒感染是由单个病毒粒子引发的，但这一结论是基于所分析的病毒基因组数量相对较少（约10-100个艾滋病毒基因组）的实验。使用uSGS，我们对急性感染中的HIV遗传学进行了深入研究，以回答以下问题：在一些似乎感染了单一病毒的个体中，是否也存在由于浅采样而遗漏的少数变体。我们能够从参加RV 254/东南亚研究合作与夏威夷010试验的15名供体中获得血浆样本（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学部分武装部队医学科学研究所逆转录病毒学部门进行的。该研究旨在确定泰国高危人群中的急性HIV感染，并立即启动ART。从急性HIV感染期间的参与者中收集了15份样本。之前有10名供体在标准SGS的急性感染中报告有一个创始人病毒，5名供体使用相同的方法显示有2到5个创始人病毒。使用我们的uSGS方法，我们证实在先前报告的10名供体中未检测到少数变异体，这些供体因单一病毒变异体的传播而感染。我们现在正在分析这些数据，以了解艾滋病毒在早期感染中的演变模式。这一目标的结果将为在急性感染中使用bNAb靶向创始病毒的研究提供信息，并将有助于我们了解艾滋病毒的传播，进化和人口动态，这些结果已在2024年逆转录病毒和病毒感染会议（CROI）上发表，并正在汇编成手稿，以便在明年出版。

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. We are also pursuing 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 performed sequencing on donor samples from pre-ART, early RAL failure, and during late RAL failure. Sequences obtained 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 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.). __ 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). Using uSGS, we performed an in-depth study of HIV genetics in acute infection, to answer the question of 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. Using our uSGS approach, we confirmed that no minority variants were detected in the 10 donors that were previously reported to have infection resulting from the transmission of a single viral variant. We are now analyzing the data to understand patterns of HIV evolution in early 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, were presented at the 2024 Conference on Retroviruses and Opportunistic Infections (CROI), and are being assembled into a manuscript for publication in the coming year.