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: 8768731
• 负责人: John Christian Tilton
• 金额: $ 27.74万
• 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/8768731
• 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; Immune response; Infection; Lymphocyte; Mass Spectrum Analysis; Measurement; Measures; Messenger RNA; Methods; Metric; Mole the mammal; Monitor; Noise; Patients; Peptide antibodies; Peptides; Population; Preparation; Proteins; Proteomics; RNA; 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; improved; instrument; instrumentation; interest; ionization; mass spectrometer; memory CD4 T lymphocyte; method development; novel; polyclonal antibody; prevent; public health relevance; 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）：潜伏感染的静息记忆 CD4+ T 细胞是根除 HIV 的主要障碍。最近，已经鉴定出许多化合物可以选择性地重新激活潜伏的艾滋病毒，这让人们燃起了通过免疫反应、抗病毒治疗或细胞病变作用重新激活和消除病毒的希望。然而，目前用于测量潜伏病毒库的“金标准”测定，即定量病毒生长测定（Q-VOA），是劳动密集型的、成本高昂的，并且需要来自多个健康捐赠者的细胞，这使得它对于大型临床试验来说不切实际。该提案旨在改进选择性反应监测质谱 (SRM-MS) 测定法，该测定法已开发用于测量患者体内潜伏 HIV 病毒库的大小。目前，我们能够在 80,000 个未感染的 CD4+ T 细胞群中检测到约 16 个受感染的细胞，这是质谱仪可以加载的最大数量。在目标 1 中，我们将评估从大量细胞（最多至少 1x107）中富集 HIV 蛋白和肽，同时检测 10 个或更少的感染细胞的策略。在目标 2 中，我们将 SRM-MS 与 HIV 感染的传统指标进行比较，包括接受抗逆转录病毒治疗至少 6 个月的病毒载量无法检测到的患者细胞中的前病毒 DNA、细胞病毒 mRNA、上清液基因组病毒 RNA 和 Q-VOA。该项目要研究的两个领域是： 1. 评估富集策略以提高 SRM-MS 的经验灵敏度。 SRM-MS 检测的理论灵敏度比单个激活的 CD4+ T 细胞估计产生的病毒低约 6 倍。 SRM-MS 的主要限制是可以加载到仪器上的蛋白质量：来自大约 80,000 个 CD4+ T 细胞的蛋白质。为了检测大量 CD4+ T 细胞中的感染细胞，我们将研究 HIV Gag 蛋白和肽富集策略。这些富集策略具有额外的优势，即在去除非 HIV 蛋白时提高我们的信噪比，从而进一步提高检测的灵敏度。 2. 比较 SRM-MS 检测与传统病毒感染检测（包括原病毒 DNA、病毒 RNA 转录本和基因组 RNA）以及使用患者样本的 Q-VOA 的灵敏度。与 Q-VOA 相比，SRM-MS 测定在通量、周转时间、样本量和成本以及潜在的灵敏度和重现性方面具有相当大的优势。为此，我们将通过与病毒感染的传统指标（包括 provirl DNA、细胞病毒 mRNA、上清液基因组病毒 RNA 和 Q-VOA）进行比较，评估 SRM-MS 测定的经验灵敏度和重现性是否足以用于检测接受抗逆转录病毒治疗时病毒载量无法检测到的患者中的潜伏 HIV。成功完成这些目标可以提供一种新颖、灵敏、高通量且经济的检测方法，用于测量参加大型临床试验的患者的潜在病毒库。