Nanopore-Based HIV Self-Test for Ultrasensitive p24 Quantification in FingerPrick Blood

摘要 尽管在艾滋病毒/艾滋病治疗方面取得了巨大进展，艾滋病毒发病率有所下降， 人口继续增长。预防艾滋病毒传播的进展仍然太慢。据估计 20%的新艾滋病毒感染是由于不知情的感染者传播。因此，早期检测 艾滋病毒对降低传播率特别重要。为此，将测试可访问性扩展到 通过自我测试的临床设置是非常需要的。艾滋病毒自我检测是一个过程，在这个过程中， 想知道他/她的艾滋病毒状况的人收集样本，进行测试，并私下解释结果。 目前的艾滋病毒自我检测技术包括快速蛋白质检测和核酸检测。次优灵敏度 目前的蛋白质检测只能支持抗体检测，并将错过很大一部分急性感染。 虽然核酸检测可以通过PCR等扩增技术达到检测下限， 会错过抗原提供的信息目前大多数关于艾滋病毒自我检测的研究工作都集中在 进行核酸检测然而，没有证据表明HIV RNA先于抗原出现。主要 抗原检测的技术挑战是蛋白质不能像核酸那样被扩增， 普遍认为抗原检测相对不敏感，因此临床应用有限。 我们先前展示了用于超灵敏抗原的点击化学放大纳米孔（CAN）测定方法， 量化该方法对HIV-1 p24抗原的检测限达到0.5 pg/ml，证明了可靠性。 在来自核酸和/或ELISA测定遗漏的患者的临床样品中检测。定量p24结果 从该方法还表明p24和病毒载量之间的相关性，提示用于监测的潜在用途 坚持抗逆转录病毒治疗，以尽量减少治疗失败。基于CAN分析，本项目旨在 开发一种超灵敏的定量HIV-1 p24抗原自我检测，以提高急性感染的早期检测 以及监测治疗效果。该测试将基于一个精简的自动设备，包括成本- 用于样品制备的有效微流控芯片和用于外行测试自己的纳米孔读取器， 指尖的血R61阶段将开发一种自测设备，用于在≤ 2 pg/ml，以支持检测急性感染和治疗失败。R33阶段将执行 在Prisma Health免疫学中心进行临床评价，以评估性能、可用性和 自检设备的可接受性。通过点击化学放大纳米孔检测的创新， 微流控样品制备，我们预计该测试将能够定量HIV-1 p24抗原低至 0.5 pg/ml，并建立p24和病毒载量水平之间的相关性。 如果成功开发和验证，这种CAN自我检测设备应该能够进行常规HIV自我检测， 简单如在家中进行血糖测试，以支持全球艾滋病毒诊断和治疗工作。

ABSTRACT Despite tremendous advances in treatment of HIV/AIDS and the decrease of HIV incidence, the overall infected population continues to grow. Progress on prevention of HIV transmission remains far too slow. It is estimated that 20% of new HIV infections are due to transmission from unaware infected individuals. Hence, early detection of HIV is particularly important for lowering transmission rates. To this end, extending testing accessibility beyond clinical settings through self-tests is highly desirable. HIV self-testing is a process in which an individual who wants to know his/her HIV status collects a specimen, performs the test, and interprets the result in private. Current HIV self-testing technologies include rapid protein tests and nucleic acid tests. The suboptimal sensitivity of current protein tests can only support antibody detection and will miss a significant portion of acute infections. Although nucleic acid tests can reach lower detection limit through amplification technologies such as PCR, they will miss the information that antigens can provide. Most current research efforts on HIV self-testing are focused on nucleic acid tests. However, there is no evidence showing that HIV RNA appears ahead of antigen. The major technological challenge for antigen detection is that proteins cannot be amplified like nucleic acids, leading to the widely held belief that antigen tests are relatively insensitive and therefore have a limited clinical utility. We previously demonstrated a click chemistry amplified nanopore (CAN) assay method for ultrasensitive antigen quantification. This assay achieved 0.5 pg/ml detection limit for HIV-1 p24 antigen and demonstrated reliable detection in clinical samples from patients missed by nucleic acid and/or ELISA assays. Quantitative p24 results from this method also indicated correlation between p24 and viral load, suggesting potential use for monitoring antiretroviral therapy adherence to minimize treatment failure. Based on the CAN assay, this project aims to develop an ultrasensitive quantitative HIV-1 p24 antigen self-test to improve early detection of acute infections and monitoring treatment efficacy. The test will be based on a streamlined automatic device including a cost- effective microfluidic chip for sample preparation and a nanopore reader for laypersons to test themselves using fingerprick blood. The R61 phase will develop a self-testing device for quantification of HIV-1 p24 antigen at ≤ 2 pg/ml in whole blood to support detection of acute infection and treatment failure. The R33 phase will perform a clinical evaluation at the Prisma Health Immunology Center to assess the performance, usability, and acceptability of the self-testing device. Through innovations in click chemistry amplified nanopore detection and microfluidic sample preparation, we anticipate the test would be able to quantify HIV-1 p24 antigen at as low as 0.5 pg/ml directly from 100µl or less finger prick blood and establish correlation between p24 and viral load levels. If successfully developed and validated, this CAN self-testing device should enable routine HIV self-testing as simple as a blood sugar test at home to support global HIV diagnostic and therapeutic efforts.