HIV-1 Genetic Variation in Infected Individuals

感染者中的 HIV-1 遗传变异

• 批准号: 8937856
• 负责人: Frank Maldarelli
• 金额: $ 71.65万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8937856
• 关键词: AIDS Vaccines; AIDS clinical trial group; Acute; Algorithms; Award; Bioinformatics; Biological Assay; Cells; Clinic; Clinical; Cloning; Collaborations; Data; Databases; Development; Distant; Drug resistance; Enrollment; Epidemic; Epidemiology; Funding; Gagging; Genetic; Genetic Epistasis; Genetic Recombination; Genetic Variation; Genotype; Goals; Gold; HIV; HIV-1; Immunology; Incidence; Individual; Infection; Integrase; Integrase Inhibitors; Investigation; Jordan; Laboratories; Lamivudine; Life; Massive Parallel Sequencing; Mediating; Methodology; Methods; Molds; Mutation; Nature; Nucleic Acids; Patients; Peripheral; Phylogenetic Analysis; Plasma; Population; Population Dynamics; Population Genetics; Population Heterogeneity; Positioning Attribute; Procedures; Process; Protocols documentation; Quality Control; Relative (related person); Reporting; Research; Resistance; Resistance development; Retrovirology; Role; Sampling; Site; Site Visit; Source; Structure; Techniques; Technology; Testing; Time; Translational Research; United States National Institutes of Health; Variant; Viral; Viremia; Virus; Virus Diseases; Zidovudine; antiretroviral therapy; assay development; base; bench to bedside; cell growth; cohort; digital; genetic analysis; genome sequencing; in vivo; insight; instrument; new technology; next generation sequencing; non-nucleoside reverse transcriptase inhibitors; population based; prevent; programs; pyrosequencing; resistance mechanism; resistance mutation; technique development; virus genetics

We are using the single-genome sequencing (SGS) technology we developed previously to analyze and understand the accumulation of genetic variation in gag/pol and env. We have made significant advances in additional assay development and have extended studies to a number of different patient groups, including chronically infected patients, both naive and on therapy, as well as in primary and early human immunodeficiency virus (HIV) infection (in collaboration with J. Margolick, E. Daar, and S. Kottilil), and in long-term nonprogressors (in collaboration with Mens). As a result, we are obtaining a more comprehensive picture of HIV genetic variation in vivo in the presence or absence of drug resistance. We have expanded analytic approaches to HIV population genetics using SGS and we have developed new technologies. The SGS approach, as developed in the DRP, is rapidly becoming the standard approach to investigate HIV populations, with a number of groups and large networks employing the technique, notably the Center for HIV/AIDS Vaccine Immunology (CHAVI). We continue to investigate the utility of the approach, and expand applications. We have collaborated with M. Jordan to compare HIV population structure as determined by SGS or standard cloning methodologies. The results demonstrate concordance between methods but also identify certain discrepancies requiring additional study. We have also collaborated with W.-S. Hu in an in-depth investigation of intersubtype recombination, demonstrating adaptive effects at distant sites. As resistance to integrase inhibitors increases, and NIH clinics are enrolling more such patients, we are preparing to extend SGS to study the integrase sequence as well. The DRP is also developing new technologies to investigate HIV-1 genetic variation. With the Translational Research Unit we have investigated the use of investigating massively parallel pyrosequencing techniques (to study HIV population genetics. Although such ultra-deep technology has been used to study HIV-1, the utility of the approach remains uncertain, because it is not clear whether the approach can accommodate a highly genetically diverse virus population and yield accurate phylogenetic data. The DRP has an extensive database of single-genome sequences from a large cohort of well-characterized patients. These single-genome sequences will provide the gold standard to compare results of pyrosequencing and determine the utility of massively parallel sequencing in genetic analysis of HIV-1 populations. We have also developed useful quality control procedures. In initial studies, we identified improvements that are essential to prevent assay-induced recombination; these optimization procedures enable pyrosequencing to be used reliably to investigate recombination and epistasis in genetically diverse populations. We are also investigating the use additional next generation sequencing approaches, including Illumina technology to obtain fine structure analysis of HIV populations in vivo. In efforts to expand our capability to perform next generation sequencing, we have obtained additional IATAP funds to acquire digital droplet PCR instrument, which will expand our capability for quantification of viral nucleic acids, We are collaborating with the Translational Research Unit to adapt dd PCR technology to sequence HIV amplified in the ddPCR process. This advance will significantly expand our single genome sequencing capability. Understanding of the expansion of genetic diversity following infection from a genetically limited to a highly diverse population has useful implications for applicability in understanding the HIV epidemic. Based on our understanding of genetic variation in acute and chronically infected individuals, we developed a new bioinformatics algorithm to discriminate between recently and chronically infected individuals based exclusively on population-based commercial genotyping data. Development of this algorithm has yielded the invention report EIR #238-2009. Field testing is currently in development, and we anticipate that this technique will be of broad epidemiologic utility in investigating incidence rates of HIV-1 infection. The development of these techniques has led to new insights in HIV population dynamics in understanding the effects of antiretroviral therapy, the nature of replication in natural suppression of HIV, and population dynamics of non-subtype B HIV populations. The nature of HIV-1 populations in patients undergoing antiretroviral therapy remains uncertain, and we are conducting an extensive genetic analysis of HIV-1 before and after initiation of antiretroviral therapy (completed Protocol 97-I-0082, new Protocol 08-I-0221). These results will yield new information regarding the nature and timing of genetic bottlenecks occurring during antiretroviral therapy. Analysis of HIV-1 sequences at relatively low viremia has been limited by technical issues in amplifying the relatively few HIV-1 sequences present in plasma during therapy. We have successfully adapted the SGS procedure to obtain acceptable numbers of sequences from patients suppressed on antiretroviral therapy. In collaboration with M. Polis and D. Persaud (NIH Bench to Bedside Award, 2006), we are analyzing genetic variation in patients enrolled in Protocol 97-I-0082 (now 08-I-0221; F. Maldarelli, PI) who have been suppressed on antiretroviral therapy for prolonged (greater than 8 y) periods. Initial analyses demonstrate that HIV does not undergo a genetic bottleneck upon initiation of antiretroviral therapy; despite 100-10,000 fold decline in levels of peripheral viremia, no significant decreases in genetic diversity were detected in the first 1-2 y of therapy. These data indicate common source of virus infecting short lived cells (responsible for greater than 90-99% of virus produced prior to therapy) and longer lived cells (responsible for virus produced 1-2 years after therapy is initiated). After prolonged therapy, emergence of predominant clones (as previously noted by Bailey et al.) was detected in the majority (7/8) patients. These data suggest that the non-clonal populations slowly decayed over time or that the clonal population increased by cellular expansion. We are also applying population genetics approaches to quantify the emergence of drug resistance mutations in rebound viremia in patients undergoing antiretroviral therapy. We are specifically investigating the relative roles of mutation and selection in development of resistance to AZT and NNRTI, as well as quantifying the role of APOBEC mediated mutations in the emergence of the M184I mutation conferring resistance to 3TC, FTC, and ddI. We are collaborating with A. Pau and H.C. Lane on a new study of HIV drug resistance in individuals undergoing antiretroviral therapy. We will be using the laboratory techniques and analytic approaches we have developed within the DRP to investigate mechanisms of resistance in clinical samples. We are collaborating with M. Kearney and J. Mellors to conduct an indepth analysis of HIV population genetics using sequencing data they generated from the ACTG 5142. Finding from this analysis will provide new information regarding position specific genetiv variation in HIV RT. [Corresponds to Project 3 in the October 2011 site visit report of the Clinical Retrovirology Section, HIV Drug Resistance Program]

我们正在使用以前开发的单基因组测序（SGS）技术来分析和了解GAG/POL和ENV中遗传变异的积累。我们在额外的测定开发方面取得了重大进步，并将研究扩展到许多不同的患者群体，包括幼稚和治疗的长期感染患者，以及原发性和早期人类免疫缺陷病毒（HIV）感染（与J. Margolick，E。Daar和S. kottilil和S. kottilil）以及与长期非生产者（与MENSENS合作）。结果，在存在或不存在耐药性的情况下，我们获得了体内HIV遗传变异的更全面的情况。我们使用SGS扩展了针对HIV人群遗传学的分析方法，并开发了新技术。在DRP中开发的SGS方法正在迅速成为研究HIV种群的标准方法，其中许多组和大型网络采用了该技术，尤其是HIV/AIDS疫苗免疫学中心（CHAVI）。我们继续研究该方法的效用，并扩展应用程序。我们已经与乔丹M.合作，比较了由SGS或标准克隆方法确定的HIV人群结构。结果表明方法之间的一致性，但也确定了需要额外研究的某些差异。我们还与W.-S.合作。 HU在深入研究subtype重组的深入研究中，表明远处的位点适应性作用。随着对整合酶抑制剂的耐药性增加，NIH诊所正在招募更多此类患者，我们正准备扩大SGS以研究整合酶序列。 DRP还正在开发新技术来研究HIV-1遗传变异。借助转化研究单元，我们已经调查了研究大量平行的焦磷酸测序技术（研究HIV人群遗传学。尽管这种超深技术已用于研究HIV-1，但该方法的实用性仍然不确定，因为该方法尚不确定，因为该方法不清楚高度依赖于高度遗传的病毒群体和单个数据序列。这些单基因组序列的特征序列将提供焦磷酸测序的结果，并在HIV-1种群的遗传分析中进行大规模平行的测序。遗传多样化的人群。我们还正在研究使用其他下一代测序方法，包括Illumina技术，以获取体内HIV群体的精细结构分析。为了扩大我们执行下一代测序的能力，我们获得了额外的IATAP资金来获取数字液滴PCR仪器，这将扩大我们对量化病毒核酸的能力，我们正在与转化研究单元合作以适应DD PCR技术，以在DDPCR过程中对HIV序列进行序列。这一进步将大大扩展我们的单个基因组测序能力。了解从遗传学限制到高度多样化人群的遗传多样性的扩展对理解HIV流行的适用性具有有用的意义。基于我们对急性和长期感染个体的遗传变异的理解，我们开发了一种新的生物信息学算法，以区分最近和长期感染的个体，仅基于基于人群的商业基因分型数据。该算法的开发产生了发明报告EIR＃238-2009。现场测试目前正在开发中，我们预计该技术在研究HIV-1感染的发病率方面将具有广泛的流行病学效用。这些技术的开发导致了HIV种群动态的新见解，了解抗逆转录病毒疗法的影响，自然抑制HIV中复制的性质以及非育型B HIV种群的种群动态。接受抗逆转录病毒疗法的患者中HIV-1种群的性质尚不确定，我们正在启动抗逆转录病毒治疗之前和之后对HIV-1进行广泛的遗传分析（完成的方案97-I-ii82，新方案08-I-I-0221）。这些结果将产生有关在抗逆转录病毒治疗期间发生的遗传瓶颈的性质和时机的新信息。在相对较低的病毒血症下对HIV-1序列的分析受到技术问题的限制，从而在治疗过程中放大血浆中相对较少的HIV-1序列。我们已经成功调整了SGS程序，以从抑制抗逆转录病毒治疗的患者中获得可接受的序列。与M. Polis和D. Persaud（2006年NIH替补席上颁奖典礼）合作，我们正在分析参与第97-I-i-ii82方案的患者的遗传变异（现为08-I-I-0221; F. Maldarelli，pi），他们在抗逆转录病毒治疗疗法方面已被抑制了（超过8 Y）。初步分析表明，艾滋病毒在开始抗逆转录病毒治疗后不会经历遗传瓶颈。尽管外周病毒血症水平下降了100-10,000倍，但在治疗的前1-2 Y中仍未检测到遗传多样性的显着降低。这些数据表明，感染短活细胞的病毒的常见来源（在治疗前产生的病毒占90-99％）和更长的细胞（负责在治疗后1 - 2年产生的病毒）。长时间治疗后，在大多数患者（7/8）患者中检测到了主要克隆的出现（如Bailey等人先前所述）。这些数据表明，非克隆人群随着时间的流逝而缓慢衰减，或者克隆人群通过细胞膨胀而增加。我们还采用种群遗传学方法来量化接受抗逆转录病毒疗法的患者反弹病毒血症中耐药性突变的出现。我们正在专门研究突变和选择在对AZT和NNRTI抗性发展中的相对作用，并量化了APOBEC介导的突变在M184i突变出现中赋予3TC，FTC和DDI的耐药性的作用。我们正在与A. Pau和H.C.合作在接受抗逆转录病毒疗法的个体中对HIV耐药性的新研究。我们将使用DRP中开发的实验室技术和分析方法来研究临床样品中抗性的机制。我们正在与M. Kearney和J. Mellors合作，使用它们从ACTG 5142产生的测序数据对HIV人群遗传学进行了深入分析。从该分析中发现将提供有关HIV RT中特定遗传学变化的新信息。 [对应于2011年10月的项目3临床逆转录病毒学部分，艾滋病毒耐药性计划的报告报告]