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）：潜伏感染的静息记忆CD4+ T细胞是根除HIV的主要障碍。最近，一些化合物已经被确定可以选择性地重新激活潜伏的HIV，这增加了病毒可以通过免疫反应、抗病毒治疗或细胞病变作用被重新激活和消除的希望。然而，目前用于测量潜伏库的“金标准”测定法，定量病毒生长测定法（Q-VOA），是劳动密集型的，昂贵的，并且需要来自多个健康供体的细胞，使其不适合大型临床试验。该提案旨在改进选择性反应监测-质谱（SRM- MS）测定，该测定已开发用于测量患者潜伏HIV库的大小。我们目前能够在80,000个未感染的CD4+ T细胞群中检测到16个感染细胞，这是质谱仪可以装载的最大数量。在Aim 1中，我们将评估从大量细胞（至少1x107）中富集HIV蛋白和肽的策略，同时检测10个或更少的感染细胞。在Aim 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、上清基因组病毒RNA，以及接受抗逆转录病毒治疗至少6个月的病毒载量检测不到的患者细胞中的Q-VOA。本项目主要研究的两个方面是：1。评价富集策略以提高SRM-MS的经验灵敏度。SRM-MS试验的理论灵敏度比单个活化CD4+ T细胞产生的病毒估计低6倍。我们对SRM-MS的主要限制是可以装载到仪器上的蛋白质数量：来自大约80,000个CD4+ T细胞的蛋白质。为了在更大的CD4+ T细胞群体中检测感染细胞，我们将研究HIV Gag蛋白和肽富集策略。这些富集策略还有一个额外的优点，即当非hiv蛋白被去除时，提高了我们的信噪比，进一步提高了检测的灵敏度。2. 比较SRM-MS分析与传统的病毒感染测量方法的敏感性，包括前病毒DNA、病毒RNA转录物和基因组RNA，以及使用患者样本的Q-VOA。与Q-VOA相比，SRM- MS分析在吞吐量、周转时间、样本量和成本以及潜在的灵敏度和重复性方面具有相当大的优势。在这个目的中，我们将通过将SRM-MS分析与传统的病毒感染指标（包括provirl DNA、细胞病毒mRNA、上清基因组病毒RNA和Q-VOA）进行比较，评估SRM-MS分析的经验敏感性和重复性是否足以用于检测在抗逆转录病毒治疗期间无法检测到病毒载量的患者的潜伏性HIV。成功完成这些目标可以提供一种新的、灵敏的、高通量的、经济的方法来测量参与大型临床试验的患者的潜在库。