Viral/Cell Determinants of HIV Drug Resistance in CNS

描述（由申请人提供）：本申请针对具有高度临床意义的关键问题。在我们的模型的基础上，我们试图确定(1)响应高活性抗逆转录病毒治疗（HAART）而产生的脑实质内HIV蛋白酶和逆转录酶突变的程度和性质，以及它们产生的机制；(2)脑脊液（CSF）中HIV的来源；包括药物敏感反弹病毒的来源，以及(3)大脑向外周播撒耐药或敏感病毒的可能性。为了解决这些问题，我们建议从接受多种抗逆转录病毒药物治疗的患者身上收集的死后骨髓和淋巴结组织，以及大脑的多个区域，包括脑膜和脉络膜丛，对一个1.2kb的HIV基因组区域进行分子克隆和测序。该区域包括整个蛋白酶基因和逆转录酶（RT）基因的那一部分，其中包含大多数已知的RT抗性突变。从相同样本的RNA （cDNA）中恢复的序列也将被检查并用于(1)解决HIV潜伏期问题，特别是巨噬细胞中的潜伏期，以及(2)确定正在进行的病毒复制的位点。为了确定脑脊液中HIV物种与循环白细胞中HIV物种之间的时间关系，我们还将从接受HAART治疗的神经系统疾病患者的脑脊液、血浆和分离的血液单核细胞和t细胞群体中克隆和测序蛋白酶- rt基因组片段以及HIV包膜基因的V1-V4区域。在可能的情况下，将对同一患者的死前和死后标本进行检查。随着耐药性突变模式的鉴定，系统发育分析将包括鉴定患者内HIV准种之间的进化关系。鉴于我们的多器官方法专注于巨噬细胞中的HIV，这些研究的结果可能会为在缺乏抗逆转录病毒耐药性的情况下导致HIV疾病进展的关键事件以及病毒学失败（包括中枢神经系统失败）提供新的见解。最佳的治疗计划需要对这些事件背后的机制有准确的理解。

DESCRIPTION (provided by applicant): This application targets key issues of high clinical significance. Building upon our model implicating systemic monocyte activation and trafficking in initiating the development of HIV neurological disease, we seek to determine (1) the extent and nature of HIV protease and reverse transcriptase mutations within the brain parenchyma that develop in response to highly active antiretroviral therapy (HAART), as well as the mechanisms whereby they arise, (2) the sources of HIV in cerebrospinal fluid (CSF), including the sources of drug-sensitive rebound virus and (3) the likelihood that the brain can seed the periphery with drug-resistant or-sensitive virus. To address these issues, we propose to molecularly clone and sequence a 1.2kb region of the HIV genome from post- mortem bone marrow and lymph node tissues, along with multiple regions of brain, including meninges and choroid plexus, collected from patients treated with multiple antiretrovirals. This region includes the entire protease gene and that portion of the reverse transcriptase (RT) gene, which harbors most of the known RT resistance mutations. Sequences recovered from RNA (cDNA) from the same specimens will also be examined and used to (1) address issues of HIV latency, particular latency in macrophages, and (2) identify site of ongoing viral replication. Towards determining temporal relationships between the HIV species in CSF and those in circulating leukocytes, we will also clone and sequence the protease-RT genomic fragment, as well as the V1-V4 region of the HIV envelope gene, from CSF, plasma and separated populations of blood monocytes and T-cells collected longitudinally from patients with and without neurological disease, receiving HAART. When available, ante-mortem and post-mortem specimens from the same patient will be examined. Along with identification of patterns of drug resistance mutations, phylogenetic analyses will include identification of evolutionary relationships among the intra-patient HIV quasispecies. Given our multi-organ approach with a focus on HIV in macrophages, the results of these studies are likely to provide new insight into the critical events leading to HIV disease progression, as well as virologic failure, including failure in the CNS, in the absence of antiretroviral resistance. Optimal treatment planning requires an accurate understanding of the mechanisms underlying these events.