Low cost molecular assay for HIV

描述（由申请人提供）：我们建议开发一种快速、低成本、阈值的检测方法，用于检测HIV疫苗试验参与者的HIV感染。艾滋病毒感染和病毒载量通常是第三期艾滋病毒疫苗接种试验的终点。正在进行的第三期艾滋病毒疫苗试验需要一种简单、低成本的分子诊断测试，可以检测免疫参与者的艾滋病毒感染。事实上，目前的分子测试过于复杂，无法用于筛查目的，免疫分析不能区分免疫和感染，CD4细胞计数不太可能检测早期艾滋病毒感染。尽管FDA要求使用经批准的检测方法来测量病毒载量，以满足许可要求，但低成本、中等复杂性的筛选试验将极大地促进这些试验。事实上，安慰剂组和疫苗组的所有参与者都必须定期进行筛查（例如，间隔3至6个月）。我们建议开发的检测将使用我们专有的解旋酶依赖性扩增（HDA）平台以及专门设计的低成本设备，用于进行分子测试，而不会用扩增产品污染实验室。HDA类似于聚合酶链反应（PCR），它使用两个引物以指数方式扩增核酸。它与PCR的不同之处在于它完全是等温的（因此不需要昂贵的热循环器）。我们建议使用用已知浓度的HIV血清阴性志愿者的全血中加入装甲RNA HIV定量克隆生成的模拟临床样本。在RT-HDA测试之前，将从血浆中分离出装甲RNA。横向流动装置已经证明了其在分离核酸扩增产品方面的实用性，并广泛用于FDA设备数据库中列出的几种中等复杂性和clia豁免的测试。扩增子的检测将通过与抗体结合的乳胶颗粒来完成，抗体结合到用于检测HDA反应产物的探针上。该试验使用三明治格式检测半抗原（生物素，FITC和地高辛）结合到一个HDA引物和每个检测探针中。用于这些检测的横向流动条有两个捕获区，因此可以检测一种分析物（HIV）以及竞争性内部控制。未在侧流装置窗口中给出条带的检测被标记为无效，而仅在控制线中具有条带的检测被标记为真阴性。在对照线和检测线位置都有条带的分析在阈值处被评分为阳性（通常是50份分析物）。在测试线上有强条带但没有控制线的分析被评为强阳性（通常超过5000份分析物）。我们将在范德比尔特大学用临床标本测试HIV检测的特异性和敏感性。试剂盒将提供给唐义伟博士（范德比尔特大学），以便他的实验室对检测结果进行评估。我们还可能向想要尝试该测试的美国政府实验室提供试剂盒。我们还将与安索拉根签订合同，为我们的竞争性内部控制设计一种装甲RNA的克隆。最后，我们将比较HDA和Roche Amplicor HIV-1 DNA PCR（版本1.5）的性能，使用一组来自CDC熟练测试（PT）计划的干血斑（DBS）标本。在I期结束时，我们将获得向FDA提交预ide的初步数据，以启动临床研究，寻求监管部门批准销售使用罗氏Amplicor HIV-1 DNA PCR（版本1.5）检测作为前置设备的人类诊断检测系统。4000万人类免疫缺陷病毒（HIV）感染者中90%以上生活在第三世界国家（见www.unaids.org/bangkok2004/GAR2004_html/GAR2004_03_en.htm）。艾滋病毒疫苗是遏制发展中国家获得性免疫缺陷综合症（艾滋病）流行的成本最低的医疗解决方案。正在进行的第三期艾滋病毒疫苗试验以防止感染（或在感染仍然发生时降低病毒载量）为终点。然而，为了满足许可的要求，FDA要求我们使用批准的检测方法来测量病毒载量。这些试验需要一种简单、低成本的分子诊断测试，可以检测免疫参与者的艾滋病毒感染。目前的证据表明，只有CD4 T细胞的中央记忆池在感染的早期受到影响。由于这只占循环CD4 T细胞的不到1%，第三世界使用的现有低成本CD4监测测试不能取代病毒RNA作为检测感染的替代品。此外，快速免疫诊断分析不能区分免疫和感染，而目前的分子诊断分析对大多数发展中国家的实验室来说过于复杂和昂贵。虽然使用干血点（DBS）将样本运送到能够进行复杂分子检测的实验室提供了解决这一问题的办法，但从患者和医学伦理的角度来看，更可取的是一种简单、低成本的分子诊断检测（即，越早发现感染，患者就越早接受抗病毒药物治疗）。一旦参与者被确定为感染，血液样本可以收集和分析与高复杂性，FDA批准的病毒载量测定。BioHelix专有的解旋酶依赖扩增（HDA）平台类似于聚合酶链反应（PCR），因为它使用两种引物以指数方式扩增核酸。HDA不同于PCR，因为它完全是等温的（因此不需要昂贵的热循环器）。事实上，HDA可以在一个简单的100美元水浴中进行。为了说明这项技术在第三世界的潜力，我们目前销售HDA试剂，作为高中教学工具包的一部分（通过Carolina Biological Supply销售）。BioHelix将这种HDA技术与一种低成本（约2- 3.75美元）的一次性设备相结合，该设备专门用于进行分子测试，而不会用扩增子污染实验室。该装置使用横向流动系统来检测放大产物。横向流动装置在许多中等复杂的测试中很常见，并且在食品和药物管理局（FDA）的设备数据库中列出了clia豁免测试。我们相信这些特性的结合使我们提出的产品成为HIV疫苗试验场所的理想选择。此外，最近的证据表明，一半以上的新获得性感染是由急性感染者引起的，他们往往是血清阴性的。一种低成本的核酸筛选试验将有助于识别这些人，减少艾滋病毒的传播，并使感染艾滋病毒的艾滋病毒疫苗接种试验的参与者受益。

DESCRIPTION (provided by applicant): We propose to develop a rapid, low cost, threshold assay for the detection of HIV infection of HIV vaccination trial participants. Infection by HIV, and viral load are the usual end points for Phase III HIV vaccination trials. Ongoing Phase III HIV vaccine trials require a simple, and low cost molecular diagnostic test that can detect HIV infection in immunized participants. Indeed, current molecular tests are too complex to be used for screening purposes, immunoassays cannot distinguish between immunization and infection, and CD4 cell counts are unlikely to detect early HIV infection. Although FDA requires the use an approved assay to measure viral load in order to meet the demands for licensure, a low cost, moderate complexity, screening test will greatly facilitate these trials. Indeed all participants in both the placebo and vaccine arms must be screened periodically (e.g., at 3 to 6 month intervals). The assay we propose to develop will use our proprietary helicase dependent amplification (HDA) platform as well as a low cost device specifically designed to perform molecular tests without contaminating the laboratory with amplification products. HDA is similar to the polymerase chain reaction (PCR) in that it uses two primers to exponentially amplify nucleic acids. It is distinct from PCR in that it is entirely isothermal (and thus does not require costly thermocyclers). We propose to use simulated clinical samples generated with the Armored RNA HIV Quant clone spiked into whole blood from HIV seronegative volunteers at known concentrations. Armored RNA will be isolated from plasma prior to RT-HDA testing. Lateral flow devices have already proven their utility in separating nucleic acid amplification products, and are widely used in several moderate complexity and CLIA-waived tests listed in the FDA device database. Detection of amplicons will be accomplished by using latex particles conjugated to antibodies that bind to the probes used to detect the HDA reaction products. The assay uses a sandwich format to detect haptens (biotin, FITC and Digoxigenin) incorporated into one of the HDA primers and into each of the detection probes. The lateral flow strip used for these assays has two capture zones, and thus allows for the detection of one analyte (HIV) as well as a competitive internal control. Assays that fail to give a band in the lateral flow device window are scored as invalid, while assays with a band in the control line alone are scored as true negative. Assays with bands at both the control and test line position are scored as positives at the threshold value (typically 50 copies of the analyte). Assays with a strong band in the test line but no control line are scored as strong positives (typically over 5000 copies of analyte). We will test the specificity and sensitivity of the HIV test with clinical specimens at Vanderbilt University. Kits will be supplied to Dr. Yi-Wei Tang (Vanderbilt University) so his laboratory can evaluate the assays. We may also supply kits to US Government labs that want to try the test. We will also contract with Assuragen to engineer a clone of Armored RNA for our competitive internal control. Finally, we will compare the performance of HDA and the Roche Amplicor HIV-1 DNA PCR (version 1.5) using a set of Dried Blood Spot (DBS) specimens from the CDC's Proficiency Testing (PT) Program. By the end of Phase I, we will have preliminary data for submission of a pre-IDE to the FDA to kick-off a clinical study to seek regulatory clearance for sale of the assay system for human diagnostics using the Roche Amplicor HIV-1 DNA PCR (version 1.5) assay as a predicate device.Narrative More than 90 % of the 40 million human immunodeficiency virus (HIV)-infected persons live in Third World countries (see www.unaids.org/bangkok2004/GAR2004_html/GAR2004_03_en.htm). An HIV vaccine is the lowest cost medical solution for containing the acquired immuno deficiency syndrome (AIDS) epidemic in developing countries. Ongoing Phase III HIV vaccine trials use protection from infection (or reduced viral load when infection still occurs) as an end point. However, in order to meet the demands for licensure the FDA requires us to use an approved assay to measure viral load. These trials require a simple, and low cost molecular diagnostic test that can detect HIV infection in immunized participants. Current evidence suggests that only the central memory pool of CD4 T cells is affected very early in infection. Since this represents less than 1% of the circulating CD4 T cells, existing low cost CD4 monitoring tests used in the third world cannot replace viral RNA as a surrogate for detecting infection. Moreover, rapid immunodiagnostic assays cannot distinguish between immunization and infection, and current molecular diagnostic assays are too complex, and expensive for most developing country laboratories. Although transporting samples, using dry blood spots (DBS), to laboratories that can perform complex molecular tests offers a solution to this problem, a simple, and low cost molecular diagnostic test is preferable from the standpoint of the patient, and medical ethics (i.e., the sooner an infection is detected the sooner the patient could receive treatment with antiviral drugs). Once a participant has been identified as infected, a blood sample can be collected and analyzed with a high complexity, FDA approved viral load assay. BioHelix's proprietary helicase dependent amplification (HDA) platform is similar to the polymerase chain reaction (PCR) in that it uses two primers to exponentially amplify nucleic acids. HDA is distinct from PCR in that it is entirely isothermal (and thus does not require costly thermocyclers). Indeed, HDA can be performed in a simple $100 water bath. To illustrate the potential of this technology for the third world, we currently sell HDA reagents as part of a teaching kit for use in high schools (sold through Carolina Biological Supply). BioHelix is combining this HDA technology with a low cost (~$2-$3.75) disposable device specifically designed to perform molecular tests without contaminating the laboratory with amplicons. The device uses a lateral flow system to detect amplification products. Lateral flow devices are common in many moderately complex, and CLIA-waived tests listed on the Food and Drug Administration (FDA) device database. We believe this combination of characteristics makes our proposed product ideal for the HIV vaccine trial sites. In addition, recent evidence suggests that more than half of the newly acquired infections are caused by acutely infected individuals who are often seronegative. A low cost nucleic acid screening test will help identify those people and reduce the spread of the HIV epidemic as well as benefit participants in HIV vaccination trials who become infected with the virus.