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

基本信息

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
项目类别：
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

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

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项目摘要

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）：潜在感染的静息记忆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在的HIV，并提出希望通过免疫反应，抗病毒疗法或细胞质病作用将病毒重新激活和消除。但是，目前用于测量潜在储层，定量病毒生长测定（Q-VOA）的“金标准”测定法是劳动密集型，昂贵的，并且需要多个健康供体的细胞，这对于大型临床试验而言是不切实际的。该提案旨在改善已开发的选择性反应监测质谱法（SRM-MS）测定法，以测量患者的潜在HIV储量的大小。目前，我们能够在80,000个未感染的CD4+ T细胞中检测到约16个感染细胞，这是可以加载到质谱仪上的最大值。在AIM 1中，我们将评估来自大量细胞（至少至少1x107）的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，我们将将SRM-MS与HIV感染的传统指标进行比较，包括病毒性DNA，细胞病毒mRNA，上清液基因组病毒RNA和Q-VOA的Q-VOA，来自抗逆转录病毒治疗至少6个月的病毒载量至少6个月的细胞中的细胞中。该项目中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测定的理论灵敏度比单个活化的CD4+ T细胞产生的病毒低6倍。我们使用SRM-MS的主要局限性是可以加载到仪器上的蛋白质：大约80,000 CD4+ T细胞的蛋白质。为了检测较大的CD4+ T细胞种群中受感染的细胞，我们将研究HIV GAG蛋白和肽富集策略。这些富集策略具有改善我们的信号与噪声比，因为除去非HIV蛋白，从而进一步提高了测定的敏感性。 2。将SRM-MS分析的敏感性与传统的病毒感染措施进行比较，包括病毒DNA，病毒RNA转录本和基因组RNA以及使用患者样品的Q-VOA。与Q-VOA相比，SRM- MS分析在吞吐量，周转时间，样本量以及成本以及可能的敏感性和可重复性方面具有相当大的优势。在此目标中，我们将评估SRM-MS测定法的经验灵敏度和可重复性是否足以用于检测患有无法检测的病毒载荷患者的潜在HIV，同时通过将其与传统的病毒性感染指标进行比较，包括病毒DNA，细胞DNA，细胞性病毒mRNA，超级n natal nemnatant，超级基因和Q-Q-Q- Q--这些目标的成功完成可能会提供一种新颖，敏感，高通量和经济测定法，以测量参加大型临床试验的患者的潜在储层。