Novel Assays for Measuring Susceptibility to Inhibitors that Target HIV1 Virion A

描述（由申请人提供）：该项目的总体目标是开发新的、稳健的、高通量的检测方法来评估HIV-1 Gag和蛋白酶蛋白抑制剂的易感性，特别强调病毒粒子的组装和成熟。这些检测将基于Monogram Bioscience广泛使用的PhenoSense HIVTM和GeneSeq HIVTM耐药检测的成熟技术，即基于重组病毒和基于核酸测序的检测，最初用于评估HIV-1蛋白酶和逆转录酶抑制剂。目前，一些组装/成熟抑制剂正在临床前和临床研究中进行评估，例如bevirimat (Panacos), vivecon （Myriad Genetics）。因此，需要可靠的、高通量的检测方法来支持这类有前途的新型抗逆转录病毒药物的开发工作，并最终将其用于常规临床实践。自2000年在Monogram临床参考实验室实施和商业化以来，PhenoSense HIV和GeneSeq HIV检测已被用于表征超过15万例患者样本。此外，在过去八年中，这两种检测方法被广泛用于支持抗逆转录病毒候选药物的III期评估，既可以表征对新药的耐药性，也可以指导优化背景方案的选择。2003年，Monogram成功地采用了PhenoSense和GeneSeq技术来评估HIV-1进入抑制剂（例如恩富维肽，马拉维洛克，vivcriviroc，伊巴利珠单抗，PRO-140），今年我们完成了两项新检测的验证，以监测对整合酶抑制剂的耐药性（例如雷替格拉韦，埃利维特格拉韦）。本应用程序描述了一种简单、可靠的策略，用于开发能够评估HIV-1 Gag和蛋白酶蛋白抑制剂的PhenoSense和GeneSeq HIV耐药分析系统；特别是针对病毒组装和成熟的抑制剂。因为这些新的检测系统将包括免疫显性Gag区，它们很可能被证明对细胞免疫反应的研究有价值。这些研究可能对疫苗设计和评价有价值。公共卫生相关性：成功完成供资提案将导致开发和验证新的基因型和表型分析，从而能够表征对艾滋病毒-1组装和成熟抑制剂具有抗性的病毒，并将有助于指导艾滋病毒-1感染患者的抗逆转录病毒治疗决策。该检测还将极大地推进gag特异性CTL逃逸变异的基因型和表型特征，并将证明对改进的HIV疫苗和免疫调节疗法的设计和开发有价值。

DESCRIPTION (provided by applicant): The overall goal of this project is to develop novel, robust, high-throughput assays to evaluate the susceptibility of to inhibitors of HIV-1 Gag and protease proteins, with particular emphasis on virion assembly and maturation. These assays will be based on proven technologies underlying Monogram Bioscience's widely used PhenoSense HIVTM and GeneSeq HIVTM drug resistance tests, i.e. recombinant virus-based and nucleic acid sequencing-based assays first developed to evaluate inhibitors of HIV-1 protease and reverse transcriptase. Currently, several assembly/maturation inhibitors are undergoing evaluation in pre-clinical and clinical studies, e.g. bevirimat (Panacos), vivecon (Myriad Genetics). Consequently, reliable, high-throughput assays are needed to support the development efforts of this promising new class of antiretroviral drugs, and eventually, their use in routine clinical practice. Since their implementation in the Monogram Clinical Reference Laboratory and commercialization in 2000, the PhenoSense HIV and GeneSeq HIV assays have been used to characterize in excess of 150,000 patient samples. In addition, both assays have been used extensively to support phase III evaluations of antiretroviral drug candidates over the past eight years, both to characterize resistance to new agents and to guide the selection of optimized background regimens. In 2003, Monogram successfully adapted the PhenoSense and GeneSeq technologies to evaluate inhibitors of HIV-1 entry (e.g. enfuvirtide, maraviroc, vivcriviroc, ibalizumab, PRO-140), and this year we completed validations for two new assays to monitor resistance to integrase inhibitors (e.g. raltegravir, elvitegravir). This application describes a straightforward, tried and true strategy to develop PhenoSense and GeneSeq HIV drug resistance assay systems that are capable of evaluating inhibitors of HIV-1 Gag and protease proteins; specifically inhibitors that target virus assembly and maturation. Because these new assay systems will include the immuno- dominant Gag region, they arelikely to prove valuable to the study of cellular immune responses. Such studies may prove valuable to vaccine design and evaluation. PUBLIC HEALTH RELEVANCE: Successful completion of the funding proposal will result in the development and validation of new genotypic and phenotypic assays that will enable the characterization of viruses that are resistant to HIV-1 assembly and maturation inhibitors and will help guide antiretroviral treatment decisions for HIV-1 infected patients. The assay will also greatly advance the genotypic and phenotypic characterization of gag-specific CTL escape variants and will prove valuable to the design and development of improved HIV vaccines and immune-modulating therapies.