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：对放射性艾滋病毒的测试策略有新的兴趣。测试潜在放射线方法的临床前模型避免了暴露于无效和潜在有害药物的暴露。此外，离体模型可能使我们能够表征为什么储层对清除具有抵抗力。我们提出了一种新方法，以评估潜在疗法对HIV离体CTL清除的影响。该模型代表了一种创新的储层表征方法，因为它使用CTL清除率作为储层蛋白表达的指标，从而使我们能够利用免疫系统的灵敏度来测量低级蛋白表达。我们的离体模型使用艺术患者的细胞研究候选抗渗透疗法的有效性：干扰素α（IFN-A）。我们发现，用IFN-A治疗后，患者来源的CD4+ T细胞显示出综合HIV DNA水平下降，随后用GAG-PRIMER的CD8+ T细胞进行共培养。使用该系统，我们将通过序列分析（AIM 1）和表达研究（AIM 2）来表征具有抗体内清除的抗病毒病毒。使用正在进行的IFN-A试验中的样品作为本金证明，我们将测试我们的离体模型是否可以预测哪些患者在体内对IFN-A治疗作出反应（AIM 3）。最后，我们将研究耐通量能够了解是否缺乏RNA或蛋白质表达可能解释缺乏间隙的前病毒克隆（AIM 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层清除率增强。我们假设可以诱导表达HIV蛋白的病毒DNA的比例（因此容易受到清除）高于先前所欣赏的。如果我们的假设是正确的，那么复制有缺陷的病毒通常是能够的。我们的评估将清除表达病毒蛋白和复制能力和有缺陷病毒的复制和有缺陷的病毒。为了检验我们的假设，我们将确定系统中表达蛋白质的细胞是否包含复制胜任病毒。值得注意的是，只要清除复制能力的病毒以及复制有缺陷的预科病毒，我们的方法就很可能具有识别有效疗法的实用性。