Detection of Latent HIV Infection Using Selective Reaction Monitoring Mass Spectr

使用选择性反应监测质谱检测潜在的 HIV 感染

• 批准号: 8874104
• 负责人: John Christian Tilton
• 金额: $ 15.85万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8874104
• 关键词: Acquired Immunodeficiency Syndrome; Anti-Retroviral Agents; Antiviral Therapy; Area; Benchmarking; Biological Assay; CD4 Positive T Lymphocytes; Cell Count; Cell Culture Techniques; Cell Volumes; Cells; Charge; Clinical Trials; DNA; Detection; Disease Progression; Economics; Enrollment; Frequencies; Funding; Gagging; Genetic Transcription; Genomics; Goals; Gold; Grant; HIV; HIV Infections; HIV-1; Health; Immune response; Infection; Lymphocyte; Mass Spectrum Analysis; Measurement; Measures; Messenger RNA; Methods; Mole the mammal; Monitor; Noise; Patients; Peptide antibodies; Peptides; Population; Preparation; Proteins; Proteomics; Raisins; Reaction; Reproducibility; Research; Rest; Sample Size; Sampling; Signal Transduction; Staging; T-Cell Activation; Techniques; Technology; Testing; Time; Transcript; Viral; Viral Load result; Viral Proteins; Virion; Virus; Virus Diseases; Work; antiretroviral therapy; assay development; base; collaboratory; cost; gag Gene Products; genomic RNA; improved; instrument; instrumentation; interest; ionization; mass spectrometer; memory CD4 T lymphocyte; method development; novel; polyclonal antibody; prevent; purge; stable isotope; tandem mass spectrometry; treatment effect; viral DNA; viral RNA

DESCRIPTION (provided by applicant): Latently infected resting memory CD4+ T cells are the primary barrier to the eradication of HIV. Recently, a number of compounds have been identified that can selectively reactivate latent HIV, raising hopes that the virus can be reactivated and eliminated through immune responses, antiviral therapy, or cytopathic effects. However, the current 'gold-standard' assay for measuring the latent reservoir, the quantitative viral outgrowth assay (Q-VOA), is labor-intensive, costly, and requires cells from multiple healthy donors, making it impractical for large clinical trials. This proposal aims to improve a selective reaction monitoring-mass spectrometry (SRM- MS) assay that has been developed to measure the size of the latent HIV reservoir in patients. We are currently able to detect ~16 infected cells among a population of 80,000 uninfected CD4+ T cells, the maximum that can be loaded onto the mass spectrometer. In Aim 1, we will evaluate strategies to enrich for HIV proteins and peptides from much larger numbers of cells (up to at least 1x107) while detecting 10 or fewer infected cells. In Aim 2, we will compare the SRM-MS with traditional metrics of HIV infection including proviral DNA, cellular viral mRNA, supernatant genomic viral RNA, and the Q-VOA in cells from patients with undetectable viral loads for at least 6 months on antiretroviral therapy. The two areas to be investigated in this project are: 1. Evaluate enrichment strategies to improve the empirical sensitivity of SRM-MS. The SRM-MS assay has a theoretical sensitivity ~6-fold lower than the virus estimated to be produced by a single activated CD4+ T cell. Our primary limitation with the SRM-MS is the amount of protein that can be loaded onto the instrument: protein from approximately 80,000 CD4+ T cells. To detect infected cells in larger populations of CD4+ T cells, we will investigate HIV Gag protein and peptide enrichment strategies. These enrichment strategies have the additional advantage of improving our signal to noise ratio as non-HIV proteins are removed, further improving the sensitivity of the assay. 2. Compare the sensitivity of the SRM-MS assay to traditional measures of viral infection including proviral DNA, viral RNA transcripts and genomic RNA, and the Q-VOA using patient samples. The SRM- MS assay has considerable advantages in throughput, turnaround time, sample size, and cost and potentially also sensitivity and reproducibility compared to the Q-VOA. In this aim, we will assess whether the empirical sensitivity and reproducibility of the SRM-MS assay are sufficient for use in detecting latent HIV in patients with undetectable viral loads while on antiretroviral therapy by comparing it with traditional metrics of viral infection including provirl DNA, cellular viral mRNA, supernatant genomic viral RNA and the Q-VOA. Successful completion of these aims could provide a novel, sensitive, high-throughput, and economic assay for measuring the latent reservoir in patients enrolled in large clinical trials.

描述(由申请人提供)：潜伏感染的静止记忆CD4+T细胞是根除艾滋病毒的主要障碍。最近，许多化合物被发现可以选择性地重新激活潜伏的艾滋病毒，这增加了人们通过免疫反应、抗病毒治疗或细胞病变效应重新激活和消除病毒的希望。然而，目前用于测量潜伏期病毒储存库的金标准分析--定量病毒生长分析(Q-VOA)--劳动密集型、费用高昂，而且需要来自多个健康捐赠者的细胞，这使得它不适用于大型临床试验。这项建议旨在改进一种选择性反应监测-质谱分析(SRM-MS)，该方法已被开发用于测量患者体内潜伏的艾滋病毒储存库的大小。我们目前能够在8万个未感染的CD4+T细胞中检测到~16个感染细胞，这是质谱仪可以加载的最大值。在目标1中，我们将评估在检测10个或更少感染细胞的同时，从更大数量的细胞(至少1×107个)中丰富HIV蛋白质和多肽的策略。在目标2中，我们将比较SRM-MS与传统的HIV感染指标，包括前病毒DNA、细胞病毒mRNA、病毒上清液RNA以及抗逆转录病毒治疗至少6个月未检测到病毒载量的患者细胞中的Q-VOA。本项目要研究的两个方面是：1.评估富集化策略以提高SRM-MS的经验灵敏度。SRM-MS检测的理论灵敏度比估计由单个激活的CD4+T细胞产生的病毒低约6倍。我们对SRM-MS的主要限制是可以加载到仪器上的蛋白质的数量：来自大约80,000个CD4+T细胞的蛋白质。为了检测更大数量的CD4+T细胞中的感染细胞，我们将研究HIV Gag蛋白和多肽浓缩策略。这些浓缩策略还有一个额外的优势，即随着非HIV蛋白的去除，我们的信噪比也会提高，从而进一步提高检测的灵敏度。2.比较SRM-MS方法与传统病毒感染检测方法的敏感性，包括病毒前病毒DNA、病毒RNA转录本和基因组RNA，以及使用患者样本的Q-VOA。与Q-VOA相比，SRM-MS在吞吐量、周转时间、样品大小和成本方面具有相当大的优势，并且具有潜在的灵敏度和重复性。为此，我们将评估SRM-MS分析的经验灵敏度和重复性是否足以用于在抗逆转录病毒治疗期间检测未检测到病毒载量的患者的潜伏HIV，方法是将其与传统的病毒感染指标(包括前病毒DNA、细胞病毒mRNA、上清基因组病毒RNA和Q-VOA)进行比较。这些目标的成功实现可以为大型临床试验中登记的患者提供一种新的、灵敏的、高通量的和经济的检测方法来测量潜在的储存库。