Mechanisms of HIV Resistance to CCR5 Inhibitors and Consequences for Pathogenesis

描述（由申请人提供）：病毒对当前抗逆转录病毒药物的耐药性以及这些药物对患者的副作用要求开发针对HIV病毒生命周期其他阶段的新抗逆转录病毒药物。阻断HIV包膜蛋白gp120和辅助受体CCR5之间相互作用的药物正在开发中，可能在不久的将来可用于治疗患者。与其他抗逆转录病毒药物一样，病毒对这些药物的耐药性是不可避免的，但关于病毒耐药性的机制以及耐药性对临床结果、病毒嗜性和疾病发病机制的影响知之甚少。该提案的具体目标是：(1)克隆和表征治疗失败的CCR5抑制剂患者的HIV包膜，(2)进行Env蛋白的结构-功能研究，以确定与耐药性相关的分子决定因素，以及(3)研究CCR5抑制剂耐药性对(a) HIV对其他进入抑制剂和中和抗体的易感性以及(b)宿主细胞嗜性和发病机制的改变的影响。本项目的研究设计和方法涉及从参与CCR5抑制剂试验的患者的血浆样本中克隆Env基因。葛兰素史克公司捐赠了接受阿普罗维洛克治疗的患者的样本，Steve Deeks博士提供了接受米拉罗维洛克治疗的患者的样本。简单地说，从血浆中分离RNA，反向转录成cDNA，通过PCR扩增病毒Env基因，并克隆到表达载体中进行细胞-细胞融合和假病毒感染性试验。克隆的Envs将检测对CCR5抑制剂、其他融合抑制剂和中和抗体的敏感性。结构-功能研究将用于确定对R5抑制剂产生耐药性的氨基酸取代。这些突变的后果将在病毒适应度测定和细胞系和原代细胞的嗜性改变中进行评估。CCR5抑制剂的研究和HIV对这些化合物产生耐药性的机制与HIV感染患者的临床护理直接相关，也可能通过进一步了解HIV进入来间接帮助寻求疫苗或免疫疗法。

DESCRIPTION (provided by applicant): Viral resistance to current antiretroviral medications and the side effects of these medications in patients necessitate the development of new antiretroviral agents that target additional stages of the HIV viral life cycle. Drugs that block the interaction between the HIV envelope protein gp120 and the coreceptor CCR5 are in development and will likely be available for the treatment of patients in the near future. As with other antiretroviral agents, viral resistance to these drugs is inevitable but little is known regarding the mechanisms of viral resistance and the consequences of resistance on clinical outcome, viral tropism, and disease pathogenesis. The specific aims of this proposal are to (1) clone and characterize HIV envelopes from patients on CCR5 inhibitors that have failed treatment, (2) perform structure-function studies of Env proteins to identify the molecular determinants associated with resistance, and (3) study the implications of resistance to CCR5 inhibitors on (a) susceptibility of HIV to other entry inhibitors and neutralizing antibodies and (b) alterations in host cell tropism and pathogenesis. The research design and methods for this project involve cloning Env genes from plasma samples from patients participating in CCR5 inhibitor trials. Samples from patients receiving aplaviroc have been donated by GlaxoSmithKline and samples from patients treated with miraviroc are being provided by Dr. Steve Deeks. Briefly, RNA is isolated from plasma, reverse transcribed into cDNA, and viral Env genes are amplified by PCR and cloned into expression vectors for cell-cell fusion and pseudovirus infectivity assays. Cloned Envs will be examined for sensitivity to CCR5 inhibitors, other fusion inhibitors, and neutralizing antibodies. Structure-function studies will be used to determine amino acid substitutions responsible for the development of resistance to R5 inhibitors. The consequences of these mutations will be assessed in viral fitness assays and alterations in tropism on cell lines and primary cells. The study of CCR5 inhibitors and the mechanisms by which HIV develops resistance to these compounds is directly relevant to clinical care of HIV-infected patients and may also indirectly aid in the pursuit of vaccines or immunotherapies by furthering our understanding of HIV entry.