Identification of Mulit-Drug Resistant HIV-1 Isolates

描述(由申请人提供)： 了解耐药的遗传基础对于使用基因耐药检测来帮助医生为患者选择最佳治疗方法、设计新的抗病毒药物以及监测耐药病毒的传播至关重要。我们计划从接受抗逆转录病毒治疗的人身上确定在HIV-1分离株中发展起来的蛋白酶、逆转录酶(RT)，以及可能的包膜和整合酶突变的模式。我们将使用这些模式来创建对每个药物类别具有高水平耐药性的典型感染性生物和分子克隆的面板，以帮助科学家研究耐药性的生物物理和生化基础。为了确定耐药性是否可以完全由药物治疗的分子靶点突变来解释，我们将比较直接从患者样本中获得的生物克隆和仅由生物克隆贡献治疗靶点的重组分子克隆的敏感性。我们还将比较具有常见耐药突变模式的A、C、D、F、G、A/E和A/G亚型分离株的重组和非重组生物克隆的敏感性。为了检验新发现的治疗相关RT和蛋白水解酶突变的作用，我们将比较除感兴趣位置突变外的同基因克隆对的易感性和复制。我们假设，具有高级别耐药的HIV-1分离株由具有相关但不同的耐药突变模式的克隆的复杂混合物组成。这些分离株的耐药程度可能低于组成该分离株的克隆的累积抗药性。对具有高级别耐药性的菌株只有部分有效的药物可能不能有效地挽救治疗，因为这些菌株可能含有只需要额外的一到两个突变就能对新药产生高度交叉耐药性的亚群。我们将在临床前和临床开发中检查来自高水平类耐药分离株的克隆对药物的敏感性，并将在这些药物浓度增加的情况下培养这些克隆，以选择具有高水平交叉耐药的病毒用于药物开发工作。

DESCRIPTION (provided by applicant): Understanding the genetic basis of drug resistance is essential for using genotypic drug resistance testing to help physicians select optimal therapy for their patients, for designing new antiviral drugs, and for monitoring the spread of drug-resistant viruses. We plan to identify patterns of protease, reverse transcriptase (RT), and perhaps envelope, and integrase mutations developing in HIV-1 isolates from persons receiving antiretroviral therapy. We will use these patterns to create panels of prototypical infectious biological and molecular clones with high-level resistance to each drug class to assist scientists investigating the biophysical and biochemical basis of drug resistance. To determine whether drug resistance can be explained solely by mutations in the molecular targets of drug therapy, we will compare the susceptibility of biological clones obtained directly from a patient sample with those of recombinant molecular clones in which only the target of therapy has been contributed by the biological clone. We will also compare the susceptibility of recombinant and non-recombinant biological clones from subtype A, C, D, F, G, A/E, and A/G isolates with common patterns of drug-resistance mutations. To examine the role of newly identified treatment-associated RT and protease mutations, we will compare the susceptibility and replication of pairs of clones that are isogenic except for mutation at the position of interest. We hypothesize that HIV-1 isolates with high-level class resistance consist of complex mixtures of clones with related but distinct patterns of drug-resistance mutations. The extent of drug resistance in such isolates is likely to be lower than the cumulative amount of drug resistance in the clones that comprise the isolate. Drugs that are only partially active against isolates with high-level class resistance may not be effective for salvage therapy because these isolates may harbor subpopulations that will require only one or two additional mutations to become highly cross-resistant to a new drug. We will examine the susceptibility of clones from high-level class-resistant isolates to drugs in pre-clinical and clinical development and will culture these clones in the presence of increasing concentrations of these drugs to select viruses with high level cross-resistance for use in drug development efforts.