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。建立在我们的模型，涉及系统性单核细胞活化和运输开始发展的艾滋病毒神经系统疾病，我们试图确定（1）的程度和性质的艾滋病毒蛋白酶和逆转录酶突变的脑实质内发展的高活性抗逆转录病毒治疗（HAART），以及机制，使他们出现，（2）来源的艾滋病毒在脑脊液（CSF），包括药物敏感性反弹病毒的来源和（3）大脑可以用耐药或敏感性病毒播种外周的可能性。为了解决这些问题，我们建议从死后骨髓和淋巴结组织中对HIV基因组的1.2kb区域进行分子克隆和测序，沿着大脑的多个区域，包括脑膜和脉络丛，从接受多种抗逆转录病毒药物治疗的患者中收集。该区域包括整个蛋白酶基因和逆转录酶（RT）基因的部分，其含有大多数已知的RT抗性突变。还将检查从相同标本的RNA（cDNA）中回收的序列，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）鉴定正在进行的病毒复制的位点。为了确定CSF中的HIV种类和循环白细胞中的HIV种类之间的时间关系，我们还将从CSF、血浆和从接受HAART的患有和不患有神经系统疾病的患者纵向收集的血液单核细胞和T细胞的分离群体中克隆蛋白酶-RT基因组片段以及HIV包膜基因的V1-V4区域并对其进行测序。如可用，将检查同一患者的宰前和宰后标本。沿着鉴定耐药突变模式，系统发育分析将包括鉴定患者体内HIV准种之间的进化关系。鉴于我们的多器官方法，重点是巨噬细胞中的HIV，这些研究的结果可能会提供新的见解，导致HIV疾病进展的关键事件，以及病毒学失败，包括在CNS中失败，在没有抗逆转录病毒耐药性的情况下。最佳治疗计划需要准确了解这些事件的机制。

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.