V3 loop characterization by ultradeep sequencing during CCR5 antagonist therapy

描述（由申请人提供）：为了给患有耐药病毒或对现有疗法有不良毒性的患者提供额外的治疗选择，正在开发具有强效抗逆转录病毒活性的靶向和阻断HIV-1进入细胞的新药。Vicriviroc （VCV）是一种正在研究的第二代CCR5 （R5）拮抗剂，在体外显示出对HIV-1的有效和选择性抑制，并在参加A5211 （IIb期临床试验）的HIV-1感染治疗经历的受试者中显示出24周的有效病毒学抑制。参加维里维洛克和马拉维洛克临床试验的受试者已经确定在治疗期间出现病毒共受体使用的变化。在使用R5抑制剂治疗期间，病毒准种的组成尤其相关，因为人们合理地担心CCR5的拮抗会导致病毒适应，出现显性使用CXCR4 （X4）的病毒，以及临床疾病的恶化。利用体内发展的cxcr4变体来表征cxcr4的特异性，以及准确描述治疗过程中出现的病毒准种，受到当前技术限制的阻碍。本提案的目的是评估使用一种新的超深测序技术来跟踪病毒准种对vicriviroc治疗反应的动态变化的可行性。使用ACTG 5211的受试者样本，我们将把这项技术应用于gp120的V3环的测序，gp120是在vicriviroc上经历过共受体使用变化的受试者的共受体使用的主要决定因素。我们将研究使用R5拮抗剂治疗的cxcr4变体的出现，并更准确地确定这些病毒的起源——要么是新出现的，要么是已经存在的少数X4群体的扩展。我们假设使用这种高度并行的测序系统将极大地提高测序的速度和深度，并且首次允许对少数病毒种群进行全面鉴定，这是目前可用技术无法实现的。更具体地说，我们建议：使用高度平行的454测序系统对经历趋向性转变的受试者的gp120 V3环进行测序，研究使用准种变异响应vicriviroc治疗时cxcr4比例的变化，并确定V3环序列在vicriviroc治疗期间的进化起源和种群动态。即使在没有药物治疗的情况下，HIV-1包膜的变化和从R5病毒向X4病毒的转变是发病和疾病进展的重要因素。拟议的研究将提供前所未有的丰富的序列数据，以阐明CCR5拮抗剂治疗对感染宿主内整个准种的病毒辅助受体使用和V3环序列进化的影响。这些研究将有助于更好地理解这些变化在疾病发病机制中可能发挥的作用。

DESCRIPTION (provided by applicant): In an effort to provide additional therapeutic options to patients with drug resistant virus or untoward toxicities on existing therapy, new drugs with potent antiretroviral activity are being developed that target and block HIV-1 entry into cells. Vicriviroc (VCV) is an investigational second-generation CCR5 (R5) antagonist that demonstrated potent and selective inhibition of HIV-1 in vitro and demonstrated potent virologic suppression through 24 weeks in HIV-1-infected treatment-experienced subjects enrolled in A5211, a phase IIb clinical trial. Subjects enrolled in clinical trials of vicriviroc and maraviroc have been identified with changes in viral co-receptor usage that emerge during therapy. The composition of the viral quasispecies is especially relevant during treatment with R5 inhibitors because there is legitimate concern that antagonism of CCR5 will result in viral adaptation, emergence of a dominant CXCR4 (X4)-using virus, and worsening of clinical disease. The characterization of the specific nature of CXCR4-using variants that develop in vivo and an accurate description of the viral quasispecies that emerge during therapy are hampered by current technological limitations. The purpose of this proposal is to assess the feasibility of using a novel ultra-deep sequencing technology to track dynamic shifts in the viral quasispecies in response to treatment with vicriviroc. Using subject samples from ACTG 5211, we will apply this technology to the sequencing of the V3 loop of gp120, a major determinant of co-receptor usage from subjects who have experienced changes in coreceptor usage on vicriviroc. We will investigate the emergence of CXCR4-using variants on R5 antagonist therapy and more accurately determine the origin of these viruses - either de novo emergence or expansion of a pre-existing minority X4 population. We hypothesize that the use of this highly parallel sequencing system will dramatically improve the speed and depth of sequencing and, for the first time, permit a comprehensive identification of minority viral populations that would not be possible using currently available techniques. More specifically, we propose to: sequence the V3 loop of gp120 from subjects experiencing tropism shifts using the highly parallel 454 sequencing system, investigate shifts in the proportion of CXCR4-using quasispecies variants in response to vicriviroc treatment, and determine the evolutionary origins and population dynamics of V3 loop sequences during vicriviroc treatment. Changes in HIV-1 envelope and the transition from R5 to X4 viruses even in the absence of drug therapy are important factors in pathogenesis and disease progression. The proposed studies will provide an unprecedented wealth of sequence data to illuminate the effects of CCR5 antagonist therapy on viral coreceptor usage and V3 loop sequence evolution across the entire quasispecies within an infected host. These studies will contribute to a better understanding of the role these changes may play in disease pathogenesis.