Development of a Control Kit for Latent HIV Diagnostics

 描述（由申请人提供）：健康问题在美国超过110万，全球有3550万人感染了艾滋病毒。随着高活性，或组合，抗逆转录病毒疗法（HAART/cART），艾滋病毒-艾滋病成为一种慢性疾病。不幸的是，HAART并不能治愈疾病;如果停止治疗，HIV病毒的产生会迅速反弹，导致全面的HIV-艾滋病。HAART不能根除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几乎没有影响，并且它不能完全控制中枢神经系统（CNS）、口腔和肠粘膜中的HIV复制。当前解决方案的问题以及产品如何满足未满足的需求最近的NIAID合同资助扩大提供Q-VOA的实验室，以评估激活或根除HAART患者中潜伏的HIV感染宿主的新疗法，并验证“潜伏和持续HIV感染的新检测方法”。“目前公认的量化潜伏水库的方法是定量病毒生长测定（Q-VOA），这是资源和时间密集型，低估了艾滋病毒潜伏水库。NIAID Q-VOA资源合同确认迫切需要加快验证和提供更准确地定量HAART患者中潜伏HIV库的检测方法。然而，目前可用的VQA和WHO HIV-1病毒标准品并不是基于细胞的HIV-1 RNA检测试剂盒的相关质控品，这在Q-VOA交叉验证过程中造成了关键瓶颈。此外，用于HIV病毒颗粒定量的Abbott和Roche RT-PCR诊断中使用的Armored RNA（r）对照试剂盒基于固有缺陷的MS 2-VLP技术。方法概述本I期提案的具体目标是开发和验证一种新型的基于海洋的病毒样颗粒（VLP），以将基于细胞的HIV-1 RNA作为潜伏HIV-1 RNA检测试验的定量标准。新型VLP HIV-1 RNA质控品将通过溶液中生物物理分析和基于HAART患者样本以及VQA和WHO HIV-1病毒标准品的RNA测定进行校准和标准化。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，校准的基于细胞的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对HAART患者中潜伏的HIV感染宿主进行高通量定量。合作者和独特的资源为了实现其第一阶段的目标，扬生物技术公司。将与美国国家标准与技术研究所（NIST）的Susan Kreuger博士、罗切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）的大卫马戈利斯博士以及VQA实验室的James Bremer博士合作。Kreuger博士是该团队的关键成员，该团队使用溶液生物物理方法来标准化病毒样颗粒（VLP）以封装RNA标准品。UR CFAR将提供HAART患者样本，用于检测VLP HIV-1 RNA标准品。Jan Biotech将与VQA实验室合作，针对目前使用的VQA和WHO HIV-1病毒标准品，对新型HIV-1 RNA VLP标准品进行并行检测。I期特异性目的特异性目的1：构建新型HIV-1 RNA病毒样颗粒（VLP）表达载体，用作潜伏HIV-1诊断试剂盒中的标准品特异性目的2：通过生物物理分析定量验证潜伏HIV RNA VLP对照特异性目的3：HIV潜伏期控制试剂盒原型的测试和分析预期结果如何证明II期和进一步产品开发的合理性I期预计将生产一种基于细胞的HIV-1 RNA VLP标准对照试剂盒，在潜伏HIV-1 RNA检测中具有已证明的实用性，并根据已建立的VQA和WHO标准进行初步验证。在第II阶段，潜伏期HIV诊断质控试剂盒将完全标准化和验证，最终目标是潜伏期HIV-1 RNA检测的标准参考物质（SRM）质控试剂盒。

 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.