Virologic Failure of boosted Proteases

增强蛋白酶的病毒学失败

• 批准号: 8525912
• 负责人: David Allenberg Katzenstein
• 金额: $ 22.14万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-07 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8525912
• 关键词: Adherence; Amino Acid Sequence; Atazanavir; Biological Assay; Cleaved cell; Clinical Treatment; Clinical Trials; Clinical Trials Cooperative Group; Cloning; Consensus; Consensus Sequence; Data; Detection; Development; Diagnostic; Diagnostic Trial; Drug Kinetics; Drug resistance; Evolution; Failure; Frequencies; Future; Gagging; Gene Mutation; Genes; Genetic; Genotype; HIV; Individual; Integrase Inhibitors; Investigation; Measures; Methodology; Methods; Minor; Minority; Molecular Cloning; Mutate; Mutation; Patients; Peptide Hydrolases; Peptide Sequence Determination; Pharmaceutical Preparations; Pilot Projects; Polyproteins; Population; Predisposition; Protease Gene; Protease Inhibitor; Protein Region; RNA-Directed DNA Polymerase; Randomized; Recombinant Proteins; Recombinants; Regimen; Relative (related person); Resistance; Risk; Ritonavir; Sampling; Site; Technology; Testing; Time; Ursidae Family; Variant; Virus; Virus Replication; deep sequencing; design; efavirenz; gag Gene Products; inhibitor/antagonist; innovation; mutant; non-nucleoside reverse transcriptase inhibitors; novel; novel diagnostics; novel strategies; nucleoside analog; nucleotide analog; polypeptide; public health relevance; pyrosequencing; randomized trial; recombinant virus; resistance mutation; response; screening; treatment strategy; treatment trial

DESCRIPTION (provided by applicant): The proposed study is a novel analysis of a large clinical trial which brings innovative sequencing approaches and novel investigation of gag gene mutation to determine the mechanism of drug resistance and failure among recipients of a boosted protease regimen, Atazanavir and ritonavir (ATV/r). We seek to understand the mechanism(s) of virologic failure among Atazanavir/ritonavir (ATV/r) recipients where we have clear evidence that adherence and pharmacokinetics are insufficient to explain virologic failure without consensus protease inhibitor mutations. The hypotheses to be explored include the investigation of minority quasispecies through cloning and ultra deep sequencing of protease to identify low levels of Protease inhibitor drug resistance mutations in the the gag gene. Alternatively, the protease and gag and gag cleavage sites (where protease cleaves the polyprotein products of HIV replication) may mutate to compete effectively with protease inhibitors. We are proposing a pilot study to determine mechanism(s) which may explain virologic failure following a boosted PI regimen. By determining the mechanism of virologic failure it will be possible to design diagnostic sequencing to identify the optimal sequential regimen(s) likely to be effective. The virologic explanations, which would have an important impact on subsequent diagnostic and treatment strategies include 1) the presence of multiple minor and major PI drug resistance mutations at levels below those detected in consensus sequencing (< 20%). Alternatively, there may be mutations selected at protease cleavage sites as mutations in the gag-pol polypeptides which allow the wild-type protease substrates to effectively compete with the inhibitor (Atazanavir) with continued replication of wild-type virus. There are implications for future treatment in either case. If there are multiple, minority PI resistance or cleavage site mutations, uncovered, then these patients would require different drugs, or drug class to achieve virologic suppression. Additionally, we will also determine whether there are mutations in the reverse transcriptase (such as M184V, K65R, or L74V) at levels that cannot be detected by consensus genotyping (<20%) but which as minority species, jeopardize a new regimen including those with a low genetic barrier to resistance such as an NNRTI (Efavirenz) or an integrase inhibitor (Raltegravir). We hypothesize that the mechanism of failure and the selection of mutations among boosted protease inhibitor recipients will point to the most effective regimen(s) for subsequent treatment. We will bring new, sensitive and innovative methodologies to bear on these questions; Ultradeep pyrosequencing and multiple cloning methods can detect low levels of mutant viruses (< 1%). This study will provide robust data towards the development of a randomized trial of new diagnostic approaches and regimens for ATV/r failures, after one or more screening tests for minority quasi-species and/or cleavage site mutations, each of which may reduce the activity of a new drug regimen. The overall aim is to enhance drug treatment strategies by examining virologic failure samples from a large randomized trial to test each of the hypothesized mechanisms for virologic failure. Data on the selection of drug resistance through minority quasi-species PI resistance mutations, gag-pol and env cleavage site mutations or both occurring below the levels of detection of standard genotyping will be key to the development of a randomized diagnostic and treatment trial to determine the best approach to ATV/r failures.

描述（由申请人提供）：拟议的研究是对一项大型临床试验的新颖分析，该试验带来了创新的测序方法和对 gag 基因突变的新颖研究，以确定加强蛋白酶方案、阿扎那韦和利托那韦（ATV/r）。我们试图了解阿扎那韦/利托那韦 (ATV/r) 接受者病毒学失败的机制，我们有明确的证据表明，在没有共识蛋白酶抑制剂突变的情况下，依从性和药代动力学不足以解释病毒学失败。待探索的假设包括通过蛋白酶克隆和超深度测序来研究少数准种，以识别 gag 基因中低水平的蛋白酶抑制剂耐药性突变。或者，蛋白酶和gag以及gag切割位点（其中蛋白酶切割HIV复制的多蛋白产物）可能发生突变以与蛋白酶抑制剂有效竞争。我们正在提议进行一项试点研究，以确定可能解释强化 PI 方案后病毒学失败的机制。通过确定病毒学失败的机制，将有可能设计诊断测序来确定可能有效的最佳序贯治疗方案。 病毒学解释将对后续诊断和治疗策略产生重要影响，包括 1) 存在多个次要和主要 PI 耐药突变，其水平低于共识测序中检测到的水平 (< 20%)。或者，可以在蛋白酶切割位点选择突变作为gag-pol多肽中的突变，其允许野生型蛋白酶底物有效地与抑制剂(阿扎那韦)竞争，从而使野生型病毒持续复制。这两种情况都对未来的治疗产生影响。如果发现存在多种、少数 PI 耐药性或切割位点突变，那么这些患者将需要不同的药物或药物类别来实现病毒学抑制。此外，我们还将确定逆转录酶（例如 M184V、K65R 或 L74V）中是否存在突变，其水平无法通过共识基因分型检测到（<20%），但作为少数物种，这些突变会危及新的治疗方案，包括对耐药性的遗传屏障较低，例如 NNRTI（依非韦伦）或整合酶抑制剂（拉特格韦）。我们假设失败的机制和加强蛋白酶抑制剂接受者中突变的选择将为后续治疗指明最有效的方案。我们将采用新的、敏感的和创新的方法来解决这些问题；超深焦磷酸测序和多重克隆方法可以检测低水平的突变病毒（< 1%）。这项研究将为 ATV/r 失败的新诊断方法和方案的随机试验的开发提供可靠的数据，在对少数准物种和/或裂解位点突变进行一项或多项筛选测试后，每种突变都可能降低活性新的药物治疗方案。总体目标是通过检查大型随机试验的病毒学失败样本来测试每种病毒学失败的假设机制，从而增强药物治疗策略。通过少数准种 PI 抗性突变、gag-pol 和 env 裂解位点突变或两者发生在标准基因分型检测水平以下而选择耐药性的数据将是制定随机诊断和治疗试验的关键，以确定解决 ATV/r 故障的最佳方法。