New Phenotypic HIV-1 Drug Resistance Assay Validation

描述（申请人提供）：提高表型的灵敏度 用于监测艾滋病患者中HIV-1耐药性的检测方法， 早期发现耐药的次要物种。我们 开发一种新的检测方法，使用分子载体系统产生指示剂 这些细胞过度表达CD 4、CXCR 4和CCR 5基因， 允许T嗜性和M嗜性HIV-1感染 可以使用直接感染方法进行抗性测定， 患者血清。这些指示细胞，含有一个tat激活的分子 控制GFP报告基因开关，提供了检测 并在药物存在和不存在的情况下量化病毒复制。系统 允许基于感染性病毒的实际水平进行病毒载量测定 礼物它应该很容易检测到低水平的耐药病毒颗粒 在与主要野生型病毒的混合物中，因为只有抗性病毒 将在药物存在的情况下在指示细胞中复制并触发 报告基因的表达。由于不涉及PCR或克隆步骤， 该测定直接测量患者的固有耐药性 原始病毒种群新的检测方法有望成为一种简单，灵敏， 用于HIV-1耐药性监测的高容量表型检测， 适用于所有类别的抗艾滋病毒药物。第一阶段SBIR-AT-NIAID赠款 申请人要求提供资金以优化、格式化和验证新的检测方法， 根据HIV-1耐药性协作组发布的指南， (RCG)在商业化之前。这将需要研究的准确性， 灵敏度、精密度、特异性和稳定性。它将涉及 对大量特征明确的HIV-1进行了广泛的回顾性研究 来自临床研究的临床分离株（野生型和耐药菌株） 合作者和来自制药公司，诊断 公司，和国家卫生研究院和确定他们的相对敏感性 目前批准的抗逆转录病毒药物。 拟定商业应用： 本研究项目是为了验证一种显著改善的新表型 具有巨大商业市场潜力的抗HIV耐药性监测检测 在艾滋病患者中。 其应用包括通过临床参考进行测定 支持患者管理和临床试验的实验室， 用于全球分销的试剂盒，基于感染性病毒水平的病毒载量测定，以及高 用于抗HIV药物发现的通量测定。

DESCRIPTION (Provided by the applicant): Improved sensitivity of phenotypic assays for monitoring HIV-1 drug-resistance in AIDS patients, with an emphasis on early detection of drug-resistant minor species, is needed. We are developing a new assay using a molecular vector system to produce indicator cells that overexpress the genes for CD4, CXCR4, and CCR5, making them permissive to both T-tropic and M-tropic HIV-1 infection so that drug resistance assays can be performed using a direct infection approach with patient sera. These indicator cells, containing a tat-activated molecular switch controlling a GFP reporter gene, provide a sensitive mechanism to detect and quantify virus replication in the presence and absence of drugs. The system allows for viral load assays based on the actual levels of infectious virus present. It should readily detect low levels of drug-resistant virus particles in a mixture with predominantly wild-type virus, since only the resistant virus will replicate in the indicator cells in the presence of drug and trigger the expression of the reporter gene. Since PCR or cloning steps are not involved, this assay measures directly the intrinsic drug resistance of the patient's original virus population. The new assay promises to be a simple, sensitive and high-capacity phenotypic test for HIV-1 drug resistance monitoring and will be applicable to all classes of anti-HIV drugs. This Phase I SBIR-AT-NIAID grant application requests funding to optimize, format, and validate the new assay, according to guidelines issued by the HIV-1 Drug Resistance Collaborative Group (RCG), prior to commercialization. This will require studies of the accuracy, sensitivity, precision, specificity and stability of the assay. It will involve extensive retrospective studies with large numbers of well-characterized HIV-1 clinical isolates (both wild-type and resistant strains) from clinical research collaborators and from repositories at pharmaceutical companies, diagnostic companies, and the NIH and the determination of their relative susceptibilities to currently approved antiretroviral drugs. PROPOSED COMMERCIAL APPLICATION: This research project is for the validation of a significantly improved new phenotypic assay with a very large commercial market potential for anti-HIV drug resistance monitoring in AIDS patients. Its applications include performance of the assay by clinical reference laboratories in support of patient management and clinical trials, development of a diagnostic kit for worldwide distribution, viral load assays based on levels of infectious virus, and high- throughput assays for anti-HIV drug discovery.