HIV-1 Genetic Variation in Infected Individuals

感染者的 HIV-1 基因变异

• 批准号: 7733253
• 负责人: Frank Maldarelli
• 金额: $ 67.48万
• 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/7733253
• 关键词: AIDS Vaccines; Award; Binding; Bioinformatics; Biological Assay; Brazil; C-terminal; Cells; Cercopithecine Herpesvirus 1; Cholesterol; Clinical; Clinical Protocols; Collaborations; Cross-Sectional Studies; Data; Databases; Depth; Development; District of Columbia; Drug resistance; Failure; Frequencies; Gagging; Gene Frequency; Genes; Genetic; Genetic Recombination; Genetic Variation; Genotype; Goals; Gold; HIV; HIV-1; Immunology; India; Individual; Infection; Macaca; Medical center; Methods; Molds; Morbidity - disease rate; Mutation; National Cancer Institute; National Institute of Allergy and Infectious Disease; Nature; Numbers; Other Genetics; Patients; Persons; Pharmacotherapy; Phenotype; Phylogenetic Analysis; Plasma; Ploidies; Pneumocystis; Population; Population Genetics; Pravastatin; Procedures; Protocols documentation; Randomized; Reporting; Research Personnel; Resistance; Sampling; Sequence Analysis; Site Visit; Standards of Weights and Measures; TSG101 gene; Techniques; Technology; Time; Tsg101 protein; United States National Institutes of Health; Universities; Variant; Viral Proteins; Viremia; Virus; Virus Diseases; antiretroviral therapy; bench to bedside; cohort; fungus; genetic analysis; genome sequencing; in vivo; inhibitor/antagonist; mortality; new technology; novel; pathogen; pol genes; programs; response; simian human immunodeficiency virus; tool; viral RNA; virology; virus genetics; virus host interaction

We are using the single genome sequencing (SGS) technology that we developed in the previous period to analyze and understand the accumulation of genetic variation in gag/pol and env in a number of different patient groups, including chronically infected patients, both nave and on therapy, as well as in primary and early HIV infection (in collaboration with Drs. Joseph Margolick, Johns Hopkins University; Eric Daar, Harbor-UCLA Medical Center; and Shyam Kottilil, NIAID). The goals of this study are to understand the nature of the forces (mutation, selection, drift, recombination) that mold the genetic diversity of virus populations in vivo. This analysis is being further extended by developing two new sequencing applications: 1) We are applying the SGS technique to analyze the proviral DNA content of cells from patients in parallel with analysis of plasma-derived HIV, as a means of understanding the distribution of the virus population in the body, as well as the potential for recombination. 2) In collaboration with Dr. John Mellors (University of Pittsburgh), we are developing a new assay (multiple genome sequencing, or MGS) as a potentially efficient way to detect resistance mutations in virus populations at low frequency. In addition, we are developing new tools to analyze sequence information, including a novel method to classify drug resistance mutations in the phylogenetic context, which is currently in development and has been applied to understanding the spread of drug resistance in experimental SHIV infections of macaques (in collaboration with Dr. Zandrea Ambrose, University of Pittsburgh). Bioinformatic approaches are also being developed to investigate correlations between the mutations identified by SGS and standard commercially available gentotypes and phenotypes (in collaboration with Dr. Margriet VanHoutte, Virco-Tibotec). In addition, we have applied phylogenetic approaches in collaboration with Joann Mican and H. Clifford Lane (NIAID) and Henry Masur (CCMD) to investigate the presence of non-B subtypes in HIV-1 infected persons in Washington, D.C. We are obtaining a more comprehensive picture of HIV genetic variation on replication in vivo in the presence or absence of drug resistance. We are using the SGS approach to look at resistance more directly, asking, for example, (in collaboration with Dr. Mellors) whether the presence of low-frequency resistance mutations in patients switching from a failed therapy is predictive of subsequent failure. We are extending the portion of the HIV genome we are sequencing to detect novel mutations associated with RT inhibitor resistance in the C-terminal region of RT (in collaboration with Dr. Vinay Pathak, National Cancer Institute). In order to investigate the presence of mutations in non-subtype B viruses, we are collaborating with several groups around the world to obtain useful samples from patients prior to and following drug therapy (Dr. Marcelo Soares, Brazil; Dr. Sunil Arora, India). We are also investigating genetic diversity of the gag p6 gene, a viral gene product that binds to host protein TSG101 in cholesterol-laden rafts and is essential for proper virus budding. Following initial observations we made on the inconsistent effects of pravastatin on HIV viral RNA levels in vivo (cross-sectional study in collaboration with Dr. Peter Sklar, Drexel University), we are studying whether gag p6 genetic diversity and specific TSG101 genotypes are associated with viral RNA levels or response to statin therapy in a new randomized multi-center clinical protocol (NIH Protocol 06-I-0197, in collaboration with Drs. Eric Freed, Mary Carrington, Anuradha Ganesan, Nancy Crum-Clianfone, Henry Masur, and Peter Sklar). 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 (NIH Protocol 97-I-0082). 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. In the last year, the HIV Drug Resistance Program (DRP) Virology Core has successfully adapted the SGS procedure to obtain acceptable numbers of sequences from patients suppressed on antiretroviral therapy. In collaboration with Drs. Michael Polis (NIAID) and Deborah Persaud (Johns Hopkins University, NIH Bench to Bedside Award, 2006), we are analyzing genetic variation in patients enolled in NIH Protocol 97-I-0082 who have been suppressed on antiretroviral therapy for a prolonged time ( more than 7 years). 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). In addition, the SGS technique has been utilized to investigate population genetics of other pathogens, and NIH investigators (Joseph Kovacs, NIH Clinical Center) have collaborated with our Host-Virus Interaction Branch in the first demonstration of genetic evidence for recombination in Pneumocystis jeroveci, an opportunistic fungus that is responsible for substantial morbidity and mortality in HIV-infected individuals. The DRP is also developing new technologies to investigate HIV-1 genetic variation. We are investigating massively parallel pyrosequencing techniques to study HIV populations 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 and allele frequencies. 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 potential utility of massively parallel sequencing in genetic analysis of HIV-1 populations. [Corresponds to Project 2 in the April 2007 site visit report of the Host-Virus Interaction Branch, HIV Drug Resistance Program]

We are using the single genome sequencing (SGS) technology that we developed in the previous period to analyze and understand the accumulation of genetic variation in gag/pol and env in a number of different patient groups, including chronically infected patients, both nave and on therapy, as well as in primary and early HIV infection (in collaboration with Drs. Joseph Margolick, Johns Hopkins University; Eric Daar, Harbor-UCLA Medical Center; and Shyam Kottilil，Niaid）。这项研究的目标是了解塑造体内病毒种群遗传多样性的力的性质（突变，选择，漂移，重组）。通过开发两个新的测序应用进一步扩展了该分析：1）我们正在应用SGS技术来分析患者的细胞病毒DNA含量与血浆衍生的HIV分析，以了解体内病毒群体的分布，以及重新组成的潜力。 2）与约翰·梅洛斯（John Mellors）博士（匹兹堡大学）合作，我们正在开发一种新测定（多个基因组测序或MGS），作为一种潜在的有效方法来检测低频病毒群体的抗性突变。此外，我们正在开发新的工具来分析序列信息，包括一种在系统发育环境中对耐药性突变进行分类的新方法，该方法目前正在开发中，并已应用于理解马克斯猕猴的耐药性传播（与Pittsburgh大学的Zandrea Ambrose博士合作）。 还开发了生物信息学方法，以研究SGS确定的突变与标准可用的绅士型和表型之间的相关性（与Virco-Tibotec的Margriet Vanhoutte博士合作）。此外，我们已经与Joann Mican和H. Clifford Lane（Niaid）和Henry Masur（CCMD）合作采用了系统发育方法，调查了华盛顿特区HIV-1感染者中非B亚型的存在。我们正在使用SGS方法更直接地查看抗药性，例如（与Mellors博士合作），是否存在从失败治疗的患者中是否存在低频耐药性突变是否可以预测随后的失败。我们正在扩展HIV基因组的部分，我们正在测序以检测RT C末端区域中与RT抑制剂耐药性相关的新突变（与国家癌症研究所的Vinay Pathak博士合作）。为了调查非囊型B病毒中突变的存在，我们正在与世界各地的几个小组合作，在药物治疗之前和之后从患者那里获得有用的样本（巴西Marcelo Soares博士；印度Sunil Arora博士）。我们还正在研究GAG P6基因的遗传多样性，GAG P6基因是一种病毒基因产物，与胆固醇筏中的宿主蛋白TSG101结合，对于适当的病毒萌芽至关重要。 在初步观察之后，我们对体内Pravastatin对HIV病毒RNA水平的不一致作用（横截面研究与Drexel University的Peter Sklar博士合作），我们正在研究GAG P6遗传多样性和特定TSG101基因型是否与病毒RNA水平或对新的RNA疗法（New New centih clantip）相关联（N 06-I-0197，与埃里克·弗里斯（Eric Freed），玛丽·卡灵顿（Mary Carrington），阿努拉德（Anuradha）Ganesan，Nancy Crum-Clianfone，Henry Masur和Peter Sklar合作。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（NIH方案97-I-I-0082）。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。在去年，HIV耐药性计划（DRP）病毒学核心已成功适应了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Drs合作。 Michael Polis（Niaid）和Deborah Persaud（Johns Hopkins University，NIH NIH替补席上颁奖典礼，2006年），我们正在分析NIH方案97-I-i-0082中埃纳罗的遗传变异，他们在抗逆转录病毒疗法上被抑制了延长的时间（超过7年）。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）。 In addition, the SGS technique has been utilized to investigate population genetics of other pathogens, and NIH investigators (Joseph Kovacs, NIH Clinical Center) have collaborated with our Host-Virus Interaction Branch in the first demonstration of genetic evidence for recombination in Pneumocystis jeroveci, an opportunistic fungus that is responsible for substantial morbidity and mortality in HIV-infected individuals. DRP还正在开发新技术来研究HIV-1遗传变异。我们正在研究大规模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和等位基因频率。 DRP具有来自大量特征良好的患者的广泛数据库。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，大规模平行测序的潜在效用。 [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]