HIV-1 Genetic Variation in Infected Individuals

感染者的 HIV-1 基因变异

• 批准号: 8349183
• 负责人: Frank Maldarelli
• 金额: $ 76.45万
• 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/8349183
• 关键词: AIDS Vaccines; Acute; Algorithms; Award; Bioinformatics; Biological Assay; Canada; Cells; Clinic; Cloning; Collaborations; DNA; Data; Databases; Developing Countries; Development; Distant; Drug resistance; Enrollment; Epidemic; Epidemiology; Gagging; Genetic; Genetic Epistasis; Genetic Recombination; Genetic Variation; Genotype; Goals; Gold; HIV; HIV-1; Immune; Immunology; Incidence; Individual; Infection; Integrase; Integrase Inhibitors; Investigation; Jordan; Life; Methodology; Methods; Molds; Mutation; Nature; Patients; Peripheral; Phenotype; Phylogenetic Analysis; Plasma; Pneumocystis; Population; Population Dynamics; Population Genetics; Population Heterogeneity; Procedures; Process; Protocols documentation; Proviruses; Public Health; Quality Control; Reporting; Research Personnel; Resistance; Role; Sampling; Site; Site Visit; Source; Structure; Techniques; Technology; Testing; Time; United States National Institutes of Health; Viremia; Virus; Virus Diseases; antiretroviral therapy; assay development; base; bench to bedside; cell growth; cohort; genetic analysis; genome sequencing; in vivo; insight; new technology; pathogen; population based; prevent; programs; technique development; viral RNA; virus genetics; virus host interaction

-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 Margolick, Daar, and Kottilil), and in long-term nonprogressors (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. We are 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. -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. We have obtained additional support for these studies (Bench to Bedside Award 2010 with Margolick and Daar). In collaboration with J. Brooks (Public Health Inst. Canada), we are expanding these studies to investigate acute infections in Canada, and in collaboration with M. Jordan we are also in the process of expanding these studies to investigate incidence rates in developing countries. In addition, bioinformatic approaches are also being developed to investigate correlations between the mutations identified by SGS and standard commercially available genotypes and phenotypes. -We have expanded the SGS approach to investigate cell-associated HIV RNA and DNA. We have developed a new technique to determine whether individual cells are infected with one or more than one provirus (supported in part by Bench to Bedside Award, 2004). Such single cell sequencing has revealed that the majority of infected cells are infected with a single provirus. Currently we are subjecting the data to rigorous statistical analysis to estimate the rate of dual and multiple infection. As multiply infected cells are the source of phenotypically mixed viruses that form the substrate for recombination, these data will yield useful information regarding the role of recombination in the spread of new mutations, including those conferring drug resistance. -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. -The HVIB is extending the understanding of HIV-1 population genetics by investigating genetic variation in elite controllers, HIV-1 infected individuals with controlled viremia in the absence of antiretroviral therapy. In collaboration with T. Benfield, H. Mens is analyzing the HIV-1 sequences amplified from longitudinal samples obtained from elite controllers over 5-10 y. Initial analysis demonstrates evidence for ongoing HIV-1 replication despite virologic control, suggesting potent immune control in these individuals (Mens et al., 2010). -The SGS technique has been used to investigate population genetics of other pathogens, and NIH investigators (J. Kovacs) have collaborated with HVIB in the first demonstration of genetic evidence for recombination in Pneumocystis jeroveci, and new detailed studies of phylogenetic structure in Pneumocystis. [Corresponds to Project 2 in the April 2007 site visit report of the Host-Virus Interaction Branch, HIV Drug Resistance Program]

- 我们正在使用以前开发的单个基因组测序（SGS）技术来分析和了解GAG/POL和ENV中遗传变异的积累。我们已经在额外的测定开发方面取得了重大进步，并已向许多不同的患者群体进行了扩展研究，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Margolick，Daar和Kottilil合作，以及长期的非生产者（Margolick，Daar和Kottilil）。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，并且我们开发了新技术。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。我们正在研究大量平行的焦磷酸测序技术来研究HIV种群遗传学。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，因为尚不清楚该方法是否可以适应高度遗传多样的病毒群体并产生准确的系统发育数据。 DRP具有来自大量具有良好特征的患者队列的单个基因组序列的广泛数据库。这些单个基因组序列将提供黄金标准，以比较焦磷酸测序的结果，并确定在HIV-1种群的遗传分析中大规模平行测序的实用性。我们还制定了有用的质量控制程序。在最初的研究中，我们确定了预防测定诱导的重组至关重要的改进。这些优化程序使得能够可靠地使用焦磷酸测序来研究遗传多样性种群中的重组和上毒。 - 理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。我们已经为这些研究获得了额外的支持（Margolick和daar的床边奖2010）。与J. Brooks（加拿大公共卫生研究所）合作，我们正在扩大这些研究，以调查加拿大的急性感染，并与M. Jordan合作，我们还在扩大这些研究以调查发展中国家的发病率。此外，还开发了生物信息学方法来研究SGS确定的突变与标准市售基因型和表型之间的相关性。 - 我们已经扩展了SGS方法，以研究与细胞相关的HIV RNA和DNA。我们已经开发了一种新技术来确定单个细胞是否感染一个或多个病毒（部分由Bench to Bedside Award，2004年）。这种单细胞测序表明，大多数感染细胞都被单个病毒感染。目前，我们正在对数据进行严格的统计分析，以估计双重感染和多重感染的速度。由于倍数感染的细胞是形成重组底物的表型混合病毒的来源，因此这些数据将获得有关重组在新突变传播中的作用的有用信息，包括赋予药物抗性的那些。 - 这些技术的发展导致了HIV种群动力学的新见解，以理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疗法之前和之后对HIV-1进行广泛的遗传分析（完成的方案97-I-i-0082，新方案08-I-II-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，我们正在分析参与方案97-I-ii82的患者的遗传变异（现为08-i-I-0221; F. Maldarelli，PI），这些患者已被抑制在抗逆转录病毒疗法上的抗逆转录病毒治疗（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。 - HVIB通过研究精英控制器的遗传变异，在没有抗逆转录病毒治疗的情况下，HIV-1感染了受控病毒血症的个体，从而扩展了对HIV-1种群遗传学的理解。 H. Mens与T. Benfield合作，分析了从超过5-10 y的精英控制器获得的纵向样品中扩增的HIV-1序列。最初的分析表明，尽管病毒控制，但仍在进行HIV-1复制的证据，表明这些个体有效的免疫控制（Mens等，2010）。 - SGS技术已用于研究其他病原体的种群遗传学，NIH研究者（J。Kovacs）与HVIB合作，首次证明了肺炎菌的重组的遗传证据，以及对肺炎静脉体中系统发育结构的新详细研究。 [对应于2007年4月的宿主病毒互动分支的现场访问报告，艾滋病毒抗药性计划的报告2]