Development of a Control Kit for Latent HIV Diagnostics

潜伏性 HIV 诊断对照试剂盒的开发

• 批准号: 9140322
• 负责人: Janet L Huie
• 金额: $ 28.89万
• 依托单位: JAN BIOTECH, INC.
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-15 至 2017-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9140322
• 关键词: 13 year old; Acquired Immunodeficiency Syndrome; Anti-Retroviral Agents; Biological Assay; Biotechnology; Blood; CD4 Positive T Lymphocytes; Cell Line; Cells; Centers for Disease Control and Prevention (U.S.); Characteristics; Chronic; Clinical; Clinical Research; Clinical Trials; Collaborations; Contracts; DNA; Detection; Development; Diagnostic; Diagnostic tests; Disease remission; Encapsulated; Enzymes; Funding; Goals; HIV; HIV Genome; HIV Infections; HIV therapy; HIV-1; Health; Highly Active Antiretroviral Therapy; Housekeeping; Institutes; Intestinal Mucosa; Laboratories; Life; Marines; Marketing; Measurement; Measures; Methods; Monitor; National Institute of Allergy and Infectious Disease; Neuraxis; Nucleic Acids; Nucleotides; Oral mucous membrane structure; Patients; Phase; Process; Production; RNA; RNA Splicing; RNA Viruses; Reference Standards; Research; Research Personnel; Resources; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleases; Sampling; Shapes; Side; System; Technology; Test Result; Testing; Time; United States Food and Drug Administration; Universities; Validation; Variant; Viral; Viral load measurement; Virion; Virus; Virus-like particle; Work; base; biophysical analysis; biophysical techniques; collaboratory; companion diagnostics; diagnostic assay; effective therapy; expression vector; high throughput screening; inhibitor/antagonist; meetings; member; novel; novel therapeutics; novel virus; particle; physical property; product development; prototype; public health relevance; quality assurance; recombinant virus; research study; vector

 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.

 描述（由申请人提供）： 健康问题 美国有超过 110 万人，全球有 3550 万人感染艾滋病毒，通过高效或联合抗逆转录病毒治疗 (HAART/cART)，艾滋病毒/艾滋病已成为一种艾滋病毒/艾滋病。不幸的是，HAART 不能治愈艾滋病毒；如果停止治疗，HIV 病毒的产生会迅速反弹，并导致全面的 HIV 感染，因为它几乎无法根除 HIV 感染。它对携带有复制能力的 HIV 基因组副本的潜伏细胞产生影响，并且无法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 最近的一份 NIAID 合同为该项目提供资金。扩大提供 Q-VOA 的实验室，以评估激活或根除 HAART 患者中潜伏 HIV 感染储库的新疗法，并验证“潜伏和持续 HIV 感染的新检测方法”。潜伏病毒库是定量病毒生长检测 (Q-VOA)，这种方法需要大量资源和时间，并且对 HIV 潜伏病毒库的检测被低估。 NIAID Q-VOA 资源合同证实迫切需要加快更准确的检测方法的验证和可用性。然而，目前可用的 VQA 和 WHO HIV-1 病毒标准并不是基于细胞的 HIV-1 RNA 检测分析的相关对照，这造成了 Q-VOA 的关键瓶颈。此外，Abbott 和 Roche RT-PCR 诊断中用于 HIV 病毒颗粒定量的 Armored RNA(r) 对照试剂盒基于固有缺陷的 MS2-VLP 技术。该第一阶段提案旨在开发和验证一种新型海洋病毒样颗粒 (VLP)，以封装基于细胞的 HIV-1 RNA，作为潜在 HIV-1 RNA 检测分析的定量标准。 HIV-1 RNA 对照将通过溶液生物物理分析以及针对 HAART 患者样本和 VQA 和 WHO HIV-1 病毒标准的基于 RNA 的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奠定基础。生产市场就绪、基于细胞的校准的 HIV-1 RNA 外部和内部标准试剂盒，适用于实验室和临床环境中 HAART 患者中潜在 HIV 感染储库的高通量定量。为了实现第一阶段的目标，Jan Biotech, Inc. 将与美国国家标准与技术研究所 (NIST) 的 Susan Kreuger 博士、Amneris Luque 博士以及罗彻斯特大学艾滋病研究中心 (UR CFAR) 的 David 博士合作艾滋病根除研究人员合作实验室 (CARE) 的 Margolis 博士和 VQA 实验室的 James Bremer 博士是使用溶液生物物理方法来治疗艾滋病的团队的关键成员。标准化用于封装 RNA 标准品的病毒样颗粒 (VLP)。 UR CFAR 将提供用于测试 VLP HIV-1 RNA 标准品的 HAART 患者样本。 Jan Biotech 将与 VQA 实验室合作对新型病毒进行并行测试。 HIV-1 RNA VLP 标准对照目前使用的 VQA 和 WHO HIV-1 病毒标准。 第一阶段具体目标 具体目标 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 具体目标 2：通过生物物理分析对潜伏 HIV RNA VLP 对照品进行定量验证 具体目标 3：原型潜伏 HIV 对照试剂盒的测试和分析 预期结果将如何证明 II 期及进一步阶段的合理性产品开发 第一阶段预计将生产一套基于细胞的 HIV-1 RNA VLP 标准对照试剂盒，在潜在 HIV-1 RNA 测定中具有经过验证的实用性，并针对已建立的 VQA 和在第二阶段，潜伏性 HIV 诊断对照试剂盒将得到全面标准化和验证，最终目标是用于潜伏性 HIV-1 RNA 检测的标准参考物质 (SRM) 对照试剂盒。