High throughput measurement of envelope gene diversity for an HIV incidence assay

用于 HIV 发病率测定的包膜基因多样性的高通量测量

• 批准号: 8466485
• 负责人: Satish Kumar Pillai
• 金额: --
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-18 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8466485
• 关键词: AIDS prevention; Biological Assay; Blood Tests; Characteristics; Chronic; Complement; DNA; DNA Binding; Data; Disadvantaged; Distant; Dyes; Entropy; Evaluation; Feasibility Studies; Future; Genes; Genetic; Genetic Variation; Gold; HIV; HIV Infections; Healthcare Systems; Human immunodeficiency virus test; Immune response; Incidence; Infection; Kinetics; Measurement; Measures; Methods; Molecular Genetics; Patients; Performance; Pilot Projects; Plasma; Procedures; Public Health; Qualitative Methods; RNA-Directed DNA Polymerase; Reagent; Relative (related person); Resolution; Sampling; T-Cell Receptor; T-Cell Receptor Genes; Temperature; Testing; Time; Validation; Viral; Viral Envelope Gene; Virus; Weight; Width; base; cost; deep sequencing; genetic technology; improved; melting; novel strategies; public health relevance; response; virus genetics

DESCRIPTION (provided by applicant): Estimating HIV incidence is an important challenge for public health. An assay that could reliably distinguish incident (new) HIV infections from chronic ones would be invaluable. Initial approaches to this problem have focused on the qualities of the host's humoral response to the virus. Unfortunately, assays based on this principle have been technically difficult and there are concerns about their accuracy. An alternative strategy is to measure the genetic diversity of a patient's virus, with diversity expected to be low in new HIV infections. Directly sequencing viral samples is expensive; measuring sequence diversity through high-resolution melting curves is more affordable, but it is only a qualitative method, and it may be misleading in patients with multiple founder viruses. Here, we propose to adapt a low-cost assay based on DNA hybridization kinetics, called AmpliCot, to the measurement of HIV gene complexity. The first aim of the project is to adapt and simplify the existing method (used to measure T cell receptor gene complexity) to viral envelope genes. This will require the synthesis of new measurement standards and the validation of new experimental conditions. The second aim is to determine how well AmpliCot measurements correlate with viral sequence complexity, sequence diversity, and Shannon entropy (a measure of sequence complexity weighted for relative abundance), with comparison to deep sequencing as a gold standard. The third aim is to test the predictive power of AmpliCot, high-resolution melting, or a combination of the two methods, against a test panel of plasma samples from subjects with new and chronic HIV infections. Serial samples from newly-infected HIV patients can be used to test whether the assay can detect an increase in viral genetic diversity over time. The reagents, methods and preliminary data from this pilot project will be used to support an R01 application for more extensive validation of this method.

描述（由申请人提供）：估计艾滋病毒发病率是公共卫生的重要挑战。可以可靠地区分事件（新的）艾滋病毒感染和慢性感染的测定是无价的。解决此问题的最初方法集中在宿主对病毒的体液反应的质量上。不幸的是，基于这一原则的测定在技术上很困难，并且人们对其准确性感到担忧。另一种策略是衡量患者病毒的遗传多样性，预计在新的HIV感染中多样性较低。直接对病毒样品进行测序很昂贵；通过高分辨率融化曲线测量序列多样性更实惠，但它只是一种定性方法，并且在具有多种创始人病毒的患者中可能会误导它。在这里，我们建议将基于DNA杂交动力学的低成本测定（称为Amplicot）适应HIV基因复杂性的测量。该项目的第一个目的是适应和简化现有方法（用于测量T细胞受体基因复杂性）到病毒包膜基因。这将需要综合新的测量标准和新实验条件的验证。第二个目的是确定放大器测量与病毒序列复杂性，序列多样性和香农熵（相对丰度加权的序列复杂度的度量）的相关性，并将其与深度测序作为金标准相比。第三个目的是测试放大器的预测能力，高分辨率熔化或两种方法的组合，以对来自具有新的和慢性HIV感染的受试者的血浆样品进行测试面板。来自新感染的HIV患者的连续样品可用于测试该测定是否可以检测到随着时间的推移病毒遗传多样性的增加。该试点项目的试剂，方法和初步数据将用于支持R01应用程序，以更广泛地验证此方法。