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.