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)对高效抗逆转录病毒治疗(HAART)产生的脑实质内艾滋病毒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)中艾滋病毒的来源，包括药物敏感反弹病毒的来源，以及(3)大脑可以向外围植入耐药或敏感病毒的可能性。为了解决这些问题，我们建议从死后骨髓和淋巴组织以及脑的多个区域，包括脑膜和脉络丛，从接受多种抗逆转录病毒治疗的患者那里收集艾滋病毒基因组的1.2kb区域，并对其进行分子克隆和测序。这一区域包括整个蛋白酶基因和逆转录酶(RT)基因的一部分，该基因包含了大多数已知的RT耐药突变。从相同样本的RNA(CDNA)中恢复的序列也将被检测并用于(1)解决HIV潜伏期问题，特别是巨噬细胞的潜伏期，以及(2)确定正在进行的病毒复制的位置。为了确定脑脊液中HIV种类和循环白细胞中HIV种类之间的时间关系，我们还将从接受HAART的脑脊液、血浆和分离的单核细胞和T细胞的纵向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。如果有，将对同一患者的死前和死后样本进行检查。除了确定耐药突变的模式外，系统发育分析还将包括确定患者体内的艾滋病毒准种之间的进化关系。鉴于我们的多器官方法侧重于巨噬细胞中的艾滋病毒，这些研究的结果可能为导致艾滋病毒疾病进展的关键事件以及病毒学失败提供新的见解，包括在缺乏抗逆转录病毒耐药性的情况下中枢神经系统的失败。最佳的治疗计划需要对这些事件背后的机制有准确的了解。

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.