Self-test HIV diagnostics utilizing structurally novel, shark-derived binding domains

项目摘要 自1980年代艾滋病危机高峰时首次引入血清学检测以来， 诊断技术的发展使艾滋病毒阳性个体的早期检测和常规监测成为可能。然而，在这方面， 有两个领域目前的艾滋病毒诊断达不到理想，我们将试图直接解决这一问题。 首先是需要一种简单的诊断工具，允许个人自我检测急性期艾滋病毒感染。 第二个挑战是提供一种诊断工具，使以前被诊断为艾滋病毒阳性的病人能够 在ART中断后监测其病毒载量，或检查由于药物出现而导致的病毒控制丧失。 阻力鉴于这些诊断将在患者自己的家中（或其他非临床环境）使用， 便宜、坚固，并且适合在没有事先培训或专业设备的情况下使用。此外，他们应该使用生物 可以以微创方式获得的样本（例如手指针刺血滴），以鼓励频繁复检 目标人群。 为了解决上述问题，我们将整合两种现有技术，以生成敏感的家庭测试。 艾滋病诊断第一项技术是一种结构新颖的结合结构域，称为VNARs，我们从 免疫鲨鱼尽管它们的尺寸很小（12 kDa），但迄今为止产生的VNAR结构域具有与VNAR结构域相同的结合亲和力。 但本质上更耐化学和热。VNAR以独特的方式与抗原相互作用 方法，并可以针对传统抗体无法获得的表位提出;我们将利用这一事实来靶向 HIV蛋白p24和Env，使我们能够捕获和准确定量游离蛋白或完整病毒， 血我们将把这些VNAR与我们的第二项技术--可编程生物纳米芯片（p-BNC）--集成在一起。 基于检测系统，其利用多孔琼脂糖微珠作为3D诊断表面。免疫测定可以是 通过用生物分子如抗体或在这种情况下VNAR装载珠来进行，允许捕获和 生物样品中所需靶标的定量。为了在非临床环境中使用该技术，我们将 开发信用卡大小的诊断盒，其核心是基于微珠的传感器阵列， 用于递送样品、洗涤缓冲液和检测试剂的系统。这将被部署和读取的电池- 供电的手持阅读器系统，将在我们的项目开发和制造。该系统将使用成像 该系统源自智能手机，能够安全可靠地将数据上传到基于云的存储库。一起 这些技术将使我们能够提供一种廉价、可靠、灵敏和准确的诊断测试， 由个人评估他们的艾滋病毒状况或密切监测他们的病毒载量，而无需诊所就诊。

PROJECT SUMMARY Since the first serologic tests were introduced at the peak of the AIDS crisis in the 1980s, continuous improvements in diagnostic technology have enabled the earlier detection and routine monitoring of HIV positive individuals. However, there are two areas where current HIV diagnostics fall short of ideal, and which we will attempt to directly address in this proposal; the first is the need for a simple diagnostic tool that allows individuals to self‐test for acute phase HIV infections. The second challenge is the provision of a diagnostic tool that will allow patients previously diagnosed as HIV positive to monitor their viral load following ART interruption or check for loss of viral control due to the emergence of drug‐ resistance. Given these diagnostics are to be used in a patients own home (or other non‐clinical, setting), they need to be cheap, robust, and suitable for use without prior training or specialist equipment. Further, they should use a biological sample that can be obtained in a minimally invasive manner (e.g. a finger‐stick blood drop) to encourage frequent retesting in target populations. To address the above problems, we will integrate two existing technologies to generate a sensitive home‐test diagnostic for HIV. The first technology is a structurally novel binding domain, so called VNARs, that we isolate from immunized sharks. Despite their diminutive size (12kDa), the VNAR domains raised thus far have binding affinities equal to classical antibodies but are intrinsically much more chemo‐ and thermostable. VNARs interact with antigen in unique ways and can be raised against epitopes that are inaccessible to conventional antibodies; we will exploit this fact to target the HIV proteins p24 and Env, allowing us to capture and accurately quantify free proteins or whole virus in unmanipulated blood. We will integrate these VNARs with our second technology, the programmable Bio‐Nano‐Chip (p‐BNC), a microchip‐ based detection system which utilizes porous agarose microbeads as 3D diagnostic surfaces. Immunometric assays can be performed by loading the beads with biomolecules such as antibodies, or in this case VNARs, allowing the capture and quantitation of desired target(s) in biological samples. To enable use of this technology in a non‐clinical setting, we will develop credit card‐sized diagnostic cartridges that have a microbead‐based sensor array at their core, and microfluidic system for the delivery of sample, wash buffer, and detection reagent. This will be deployed and read by a battery‐ powered handheld reader system that will be developed and fabricated during our project. This system will use an imaging system derived from smartphones and enable the safe and secure uploading of data to a cloud‐based repository. Together these technologies will allow us to deliver an inexpensive, robust, sensitive, and accurate diagnostic test that can be used by an individual to assess their HIV status or closely monitor their viral load, without a clinic visit.