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

利用结构新颖的鲨鱼衍生结合域进行 HIV 自检诊断

• 批准号: 10539323
• 负责人: Helen M. Dooley
• 金额: $ 46.94万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10539323
• 关键词: 3-Dimensional; Acquired Immunodeficiency Syndrome; Acute; Address; Affinity; Animals; Antibodies; Antigen-Antibody Complex; Antigens; Area; Binding; Biological; Biological Assay; Blood; Blood specimen; Buffers; Capsid Proteins; Cellular Phone; Clinic Visits; Coupling; Data; Databases; Detection; Diagnosis; Diagnostic; Diagnostic tests; Dissociation; Drops; Drug resistance; Early Diagnosis; Environment; Epidemiology; Epitopes; Equipment; Feedback; Fingers; Generations; HIV; HIV Core Protein p24; HIV Infections; HIV Seropositivity; HIV-1; Harvest; Health Personnel; Healthcare; Home; Human; Human immunodeficiency virus test; Immunize; Individual; Interruption; Laboratories; Libraries; Link; Microfluidics; Microspheres; Monitor; Monoclonal Antibodies; Nurses; Outcome; Patients; Performance; Peripheral Blood Lymphocyte; Phase; Porosity; Privatization; Proteins; Reader; Reagent; Recombinants; Reproducibility; Resources; Risk; Sampling; Secure; Self Administration; Sepharose; Serology test; Shark; Specialist; Specificity; Surface; System; Tablets; Target Populations; Technology; Testing; Time; Training; Validation; Viral; Viral Load result; Virus; Weight; accurate diagnostics; antiretroviral therapy; assay development; cloud based; detection platform; diagnostic platform; diagnostic technologies; diagnostic tool; digital; drug resistant virus; falls; home test; imaging system; improved; microchip; minimally invasive; novel; point of care; programs; progression risk; prototype; repository; response; self testing; sensor; thermostability; virology

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.

项目概要 自 20 世纪 80 年代艾滋病危机高峰期首次推出血清学检测以来，检测技术不断改进 诊断技术的进步使得对艾滋病毒阳性者的早期检测和常规监测成为可能。 当前的艾滋病毒诊断有两个领域不够理想，我们将在本报告中尝试直接解决这两个领域 提案；第一个是需要一种简单的诊断工具，使个人能够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，使之前被诊断为艾滋病毒呈阳性的患者能够 在 ART 中断后监测其病毒载量，或检查是否因药物的出现而导致病毒控制丧失。 鉴于这些诊断要在患者自己的家中（或其他非临床环境）使用，因此它们需要被使用。 便宜、坚固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。 可以通过微创方式获取样本（例如指尖血滴）以鼓励频繁重新检测 在目标人群中。 为了解决上述问题，我们将整合两种现有技术来生成敏感的家庭测试 第一项技术是一种结构新颖的结合域，即我们从其中分离出来的 VNAR。 尽管免疫鲨鱼的尺寸很小（12kDa），但迄今为止产生的 VNAR 结构域具有相同的结合亲和力。 VNAR 与经典抗体相比具有更高的化学稳定性和热稳定性，并且以独特的方式与抗原相互作用。 我们将利用这一事实来靶向传统抗体无法接触到的表位； HIV 蛋白 p24 和 Env，使我们能够捕获并准确定量未操作的游离蛋白或整个病毒 我们将把这些 VNAR 与我们的第二种技术集成，即可编程生物纳米芯片 (p-BNC)，这是一种微芯片。 基于利用多孔琼脂糖微珠作为 3D 免疫测定表面的检测系统。 通过在珠子上装载生物分子（例如抗体，或在本例中为 VNAR）来执行，从而允许捕获和 生物样品中所需目标的定量为了能够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，其核心具有基于微珠的传感器阵列和微流体 用于输送样品、洗涤缓冲液和检测试剂的系统这将由电池部署和读取。 我们的项目将开发和制造动力手持式读取器系统，该系统将使用成像技术。 源自智能手机的系统，可以将数据安全可靠地上传到基于云的存储库。 这些技术将使我们能够提供一种廉价、稳健、灵敏且准确的诊断测试，可供使用 由个人评估其艾滋病毒状况或密切监测其病毒载量，而无需前往诊所。