An ex vivo model to predict outcomes and probe mechanism of anti-reservoir agents

预测抗储库药物结果和探讨机制的离体模型

• 批准号: 8930064
• 负责人: Una T O'Doherty
• 金额: $ 22万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-19 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8930064
• 关键词: Aftercare; Biological Assay; CD28 gene; CD3 Antigens; CD4 Positive T Lymphocytes; CD8B1 gene; Categories; Cells; Clear Cell; Clinical Trials; Coculture Techniques; Combined Modality Therapy; DNA; Data; Defective Viruses; Drops; Drug Targeting; Effectiveness; Epigenetic Process; Exposure to; Frequencies; Gagging; Genetic Transcription; HIV; HIV Antigens; Head; Immune system; In Vitro; Interferon-alpha; Measurement; Measures; Mediating; Modeling; Monitor; Outcome; Patients; Pre-Clinical Model; Process; Proteins; Proviruses; RNA; Resistance; Sampling; Sequence Analysis; System; T-Lymphocyte; Testing; Viral Proteins; Virus; Vision; base; design; effective therapy; gag Gene Products; immune clearance; in vivo; innovation; insight; interest; novel; novel strategies; pre-clinical; protein expression; public health relevance; therapy design; viral RNA

 DESCRIPTION: There is renewed interest in testing strategies to eradicate HIV. A preclinical model for testing potential eradication approaches avoids exposing patients to ineffective and potentially harmful agents. Moreover, an ex vivo model might allow us to characterize why the reservoir is resistant to clearance. We propose a new approach to evaluate the effect of potential therapies on CTL clearance of HIV ex vivo. This model represents an innovative approach to reservoir characterization because it uses CTL clearance as an indicator of reservoir protein expression, allowing us to take advantage of the sensitivity of the immune system to measure low-level protein expression. Our ex vivo model uses cells from ART patients to study the effectiveness of a candidate anti-reservoir therapy: interferon alpha (IFN-a). We find that patient-derived CD4+ T cells show a drop in levels of integrated HIV DNA after treatment with IFN-a and subsequent coculture with Gag- primed CD8+T cells. Using this system we will characterize the proviruses that are resistant to ex vivo clearance by sequence analysis (Aim 1) and expression studies (Aim 2). Using samples from an ongoing IFN-a trial as a proof of principal, we will test whether our ex vivo model can predict which patients respond to IFN-a therapy in vivo (Aim 3). Finally, we will study the proviral clones that are resistant to clearance to understand if lack of RNA or protein expression may explain lack of clearance (Aim 4). We also will determine whether repeat stimulation or combination stimulation leads to enhanced reservoir clearance in coculture. We hypothesize that the fraction of proviral DNA that can be induced to express HIV proteins (and is therefore susceptible to clearance) is higher than previously appreciated. If our hypothesis is correct, then replication defective viruses are often capable of expressing viral protein and both replication competent and defective viruses will be cleared in our assay. To test our hypothesis, we will determine whether the protein-expressing cells in our system contain replication competent proviruses. Notably, as long as replication competent viruses are cleared along with replication defective proviruses, then our approach will likely have utility in identifying effective therapies.

 描述：人们对测试根除艾滋病毒的策略重新产生了兴趣，用于测试潜在根除方法的临床前模型可以避免患者接触无效且可能有害的药物。此外，离体模型可能使我们能够描述为什么储存库难以清除。我们提出了一种新方法来评估潜在疗法对 HIV 离体 CTL 清除的影响。该模型代表了一种创新的储库表征方法，因为它使用 CTL 清除作为储库蛋白表达的指标，使我们能够利用敏感性。的我们的离体模型使用 ART 患者的细胞来研究候选抗储库疗法的有效性：干扰素 α (IFN-a)。使用 IFN-a 处理并随后与 Gag 引发的 CD8+T 细胞共培养后，整合的 HIV DNA 水平下降。使用该系统，我们将通过序列分析（目标 1）和表达来表征对离体清除有抵抗力的原病毒。研究（目标 2）。使用正在进行的 IFN-a 试验的样本作为原则证明，我们将测试我们的离体模型是否可以预测哪些患者对体内 IFN-a 治疗有反应（目标 3）。研究对清除具有抵抗力的原病毒克隆，以了解 RNA 或蛋白质表达的缺乏是否可以解释清除的缺乏（目标 4）。我们还将确定重复刺激或组合刺激是否会导致共培养中的储库清除率增强。规定的比例可以被诱导表达HIV蛋白（因此容易被清除）的DNA比之前估计的要高，如果我们的假设是正确的，那么复制缺陷型病毒通常能够表达病毒蛋白，并且复制能力和缺陷型病毒都将被清除。为了检验我们的假设，我们将检测系统中的蛋白质表达细胞是否含有具有复制能力的原病毒。值得注意的是，只要能够复制的病毒与复制缺陷型原病毒一起被清除，那么我们的方法就可能在识别方面有用。有效的疗法。