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

描述（由申请人提供）：估计艾滋病毒发病率是公共卫生的一项重要挑战。一种能够可靠地区分新发艾滋病毒感染和慢性感染的检测方法将是非常宝贵的。对这个问题的初步研究集中在宿主对病毒的体液反应的质量上。不幸的是，基于该原理的测定在技术上是困难的，并且存在对其准确性的担忧。另一种策略是测量患者病毒的遗传多样性，预计新的艾滋病毒感染者的多样性较低。直接对病毒样本进行测序是昂贵的;通过高分辨率熔解曲线测量序列多样性更经济实惠，但它只是一种定性方法，在有多种创始人病毒的患者中可能会产生误导。在这里，我们建议采用一种基于DNA杂交动力学的低成本检测方法，称为AmpliCot，以测量HIV基因的复杂性。该项目的第一个目的是适应和简化现有的方法（用于测量T细胞受体基因的复杂性），以病毒包膜基因。这将需要合成新的测量标准和验证新的实验条件。第二个目标是确定AmpliCot测量与病毒序列复杂性、序列多样性和香农熵（一种针对相对丰度加权的序列复杂性度量）的相关性，并将其与深度测序进行比较作为金标准。第三个目的是测试AmpliCot、高分辨率熔解或两种方法的组合对来自新的和慢性HIV感染受试者的血浆样本的测试组的预测能力。来自新感染的HIV患者的系列样本可用于测试该检测方法是否可以检测到病毒遗传多样性随时间的增加。该试验项目的试剂、方法和初步数据将用于支持R 01申请，以便对该方法进行更广泛的验证。

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.