Development of a Control Kit for Latent HIV Diagnostics

 描述(申请人提供)：美国的健康问题远远超过110万人，全球有3550万人感染艾滋病毒。随着高活性或联合抗逆转录病毒疗法(HAART/CART)的实施，艾滋病毒/艾滋病成为一种慢性病。不幸的是，HAART不是治愈方法；如果停止治疗，艾滋病毒产量迅速反弹，并导致艾滋病毒-艾滋病的全面爆发。HAART未能根除HIV感染，因为它对携带有复制能力的HIV基因组拷贝的潜伏细胞影响不大，而且它未能完全控制中枢神经系统(CNS)、口腔和肠道粘膜中的HIV复制。当前解决方案的问题和产品如何满足未得到满足的需求NIAID最近的一份合同为扩大提供Q-VOA的实验室提供资金，以评估激活或根除HAART患者潜在HIV感染宿主的新疗法，并验证“潜在和持续HIV感染的新分析”。目前被接受的量化潜伏库的方法是定量病毒扩展分析(Q-VOA)，这种方法是资源和时间密集型的，低估了HIV潜伏库。NIAID Q-VOA资源合同确认，迫切需要加快验证和提供更准确地量化HAART患者中潜在艾滋病毒宿主的检测方法。然而，目前可用的VQA和世卫组织HIV-1病毒标准并不是基于细胞的HIV-1 RNA检测的相关控制，这在Q-VOA交叉验证过程中造成了一个关键的瓶颈。此外，雅培和罗氏RT-PCR诊断中使用的用于HIV病毒颗粒定量的Armored RNA(R)控制试剂盒基于固有缺陷的MS2-VLP技术。方法概述这一阶段第一阶段提案的具体目标是开发和验证一种新型的海洋病毒样颗粒(VLP)，以包裹基于细胞的HIV-1RNA作为潜伏的HIV-1RNA检测的定量标准。新的VLP HIV-1 RNA对照将通过溶液中的生物物理分析以及针对HAART患者样本的基于RNA的分析以及VQA和世卫组织HIV-1病毒标准进行校准和标准化。第一阶段的结果将为第二阶段的全面验证和生产市场准备就绪的、基于校准细胞的HIV-1 RNA外部和内部标准试剂盒奠定基础，该试剂盒适用于在实验室和临床环境中高通量量化HAART患者潜在的HIV感染宿主。合作者和独特资源为了实现其第一阶段目标，Jan Biotech，Inc.将与美国国家标准与技术研究所(NIST)的Susan Kreuger博士、罗彻斯特大学艾滋病研究中心(UR CFAR)的Amneris Luque博士、根除艾滋病研究人员合作实验室(CARE)的David MarGolis博士以及VQA实验室的James Bremer博士合作。Kreuger博士是使用溶液生物物理方法标准化病毒样颗粒(VLP)以封装RNA标准的团队的关键成员。我们的CFAR将提供HAART患者样本，用于测试VLP HIV-1 RNA标准。Jan Biotech将与VQA实验室合作，对新的HIV-1 RNA VLP标准与当前使用的VQA和WHO HIV-1病毒标准进行并行测试。第一阶段具体目标1：构建新的HIV-1 RNA病毒样颗粒(VLP)表达载体，用作潜伏HIV-1诊断试剂盒的标准2：通过生物物理分析特定目标3：测试和分析原型潜在HIV控制试剂盒如何证明预期结果将证明第二阶段和进一步的产品开发是合理的第一阶段预计将生产一套以细胞为基础的HIV-1 RNA VLP标准控制试剂盒，经证明在潜伏HIV-1 RNA检测中有实用价值，并根据既定的VQA和世卫组织标准进行初步验证。在第二阶段，潜伏的艾滋病毒诊断控制试剂盒将完全标准化和验证，最终目标是用于潜伏的艾滋病毒-1核糖核酸分析的标准参考物质(SRM)控制试剂盒。

 DESCRIPTION (provided by applicant): Health Problem Well over 1.1 million in the U.S., and 35.5 million people globally, are infected with HIV. With highly active, or combination, anti-retroviral therapy (HAART/cART), HIV-AIDS becomes a chronic condition. Unfortunately, HAART is not a cure; if treatment is stopped, HIV virus production rapidly rebounds and leads to full-blown HIV-AIDS. HAART fails to eradicate HIV infection because it has little effect on latent cells carrying replication- competent HIV genome copies, and it fails to fully control HIV replication in the central nervous system (CNS), oral and intestinal mucosa. Issues with Current Solutions & How Product Meets Unmet Needs A recent NIAID contract funds the expansion of laboratories offering Q-VOA to evaluate novel therapies to activate or eradicate latent HIV-infected reservoirs in HAART patients and to validate "novel assays of latent and persistent HIV infection." The currently accepted method of quantifying the latent reservoir is the quantitative viral outgrowth assay (Q-VOA), which is resource and time intensive and underestimates the HIV latent reservoir. The NIAID Q-VOA Resource contract confirms a dire need to expedite the validation and availability of assays that more accurately quantitate latent HIV reservoirs in HAART patients. However, the currently available VQA and WHO HIV-1 virus standards are not relevant controls for cell-based HIV-1 RNA detection assays, creating a critical bottleneck in the Q-VOA cross-validation process. Further, Armored RNA(r) control kit used within the Abbott and Roche RT-PCR diagnostics for HIV virus particle quantification is based on the inherently flawed MS2-VLP technology. Summary of Approach The goals of the Specific Aims of this Phase I proposal are to develop and validate a novel, marine-based virus-like particle (VLP) to encapsidate cell-based HIV-1 RNA as quantitative standards for latent HIV-1 RNA detection assays. The novel VLP HIV-1 RNA controls will be calibrated and standardized through in solution biophysical analysis and within an RNA-based assay against HAART patient samples and the VQA and WHO HIV-1 viral standards. The Phase I results will lay the groundwork for Phase II full validation and production of a market-ready, calibrated cell-based HIV-1 RNA external and internal standards kit suitable for high- throughput quantification of latent HIV-infected reservoirs in HAART patients in laboratory and clinical settings. Collaborators and Unique Resources To accomplish its Phase I aims, Jan Biotech, Inc. will collaborate with Dr. Susan Kreuger at National Institute of Standards and Technology (NIST), Dr. Amneris Luque and University of Rochester Center for AIDS Research (UR CFAR), Dr. David Margolis of the Collaboratory of AIDS Researchers for Eradication (CARE), and Dr. James Bremer of the VQA Laboratory. Dr. Kreuger was a key member of the team that used in solution biophysical methods to standardize virus-like particle (VLP) for encapsulation of RNA standards. UR CFAR will provide HAART patient samples for testing the VLP HIV-1 RNA standards. Jan Biotech will work with the VQA Laboratory to perform side-by-side testing of the novel HIV-1 RNA VLP standards against the currently used VQA and WHO HIV-1 viral standards. Phase I Specific Aims Specific Aim 1: Construction of Novel HIV-1 RNA Virus-Like Particle (VLP) Expression Vectors for Use as Standards in a Latent HIV-1 Diagnostic Kit Specific Aim 2: Quantitative Validation of the Latent HIV RNA VLP Controls by Biophysical Analysis Specific Aim 3: Testing and Analysis of the Prototype Latent HIV Control Kit How Anticipated Results will Justify Phase II and Further Product Development The Phase I is expected to produce a kit of cell-based HIV-1 RNA VLP standard controls, with proven utility in a latent HIV-1 RNA assay and preliminary validation against the established VQA and WHO standards. In the Phase II, the latent HIV diagnostic control kit will be fully standardized and validated, with the end goal of standard reference material (SRM) control kit for latent HIV-1 RNA assays.