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

 描述：人们对根除艾滋病毒的测试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避免将患者暴露于无效和潜在有害的药剂。此外，离体模型可能使我们能够表征为什么储库对清除具有抗性。我们提出了一种新的方法来评估潜在的治疗方法对CTL清除HIV的体外效果。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，使我们能够利用免疫系统的敏感性来测量低水平的蛋白表达。我们的离体模型使用来自ART患者的细胞来研究候选抗储库疗法：干扰素α（IFN-α）的有效性。我们发现，在用IFN-α处理并随后与Gag致敏的CD 8 +T细胞共培养后，患者来源的CD 4 + T细胞显示整合的HIV DNA水平下降。使用该系统，我们将通过序列分析（目的1）和表达研究（目的2）来表征对离体清除具有抗性的前病毒。使用来自正在进行的IFN-a试验的样品作为原理的证据，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（目的3）。最后，我们将研究对清除有抗性的前病毒克隆，以了解RNA或蛋白质表达的缺乏是否可以解释清除的缺乏（目的4）。我们还将确定是否重复刺激或联合刺激导致增强水库清除共培养。我们假设，可以被诱导表达HIV蛋白（因此容易被清除）的前病毒DNA的比例高于以前的估计。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，并且在我们的测定中将清除复制能力和缺陷型病毒。为了验证我们的假设，我们将确定我们系统中的蛋白表达细胞是否含有可复制的前病毒。值得注意的是，只要有复制能力的病毒与复制缺陷的前病毒一起被沿着清除，那么我们的方法将可能在鉴定有效疗法中具有实用性。

 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.