Application and development of molecular biological methods to the issues of va

分子生物学方法在VA问题中的应用和发展

• 批准号: 6433517
• 负责人: KEITH PEDEN
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6433517
• 关键词: Retroviridae; drug quality /standard; human tissue; nucleic acid sequence; polymerase chain reaction; vaccines

The goal of this project is to apply and develop molecular biological methods that can ensure that vaccines and cell substrates are free from viral, and particularly retroviral, contamination. Three sensitive methods for the detection of reverse transcriptase (RT) that are at least a million-fold more sensitive than conventional RT assays have been published. Although the assays were variously termed PERT (for product- enhanced RT) or Amp-RT, we have chosen to call them by the generic name of PBRT for PCR-based RT. The three assays depend on the same principle: an RNA of known sequence is used as a template with an oligodeoxynucleotide primer in an RT reaction; because the sequence of the RNA is known, the cDNA product can be amplified by the polymerase chain reaction (PCR); the PCR product can be detected by a number of methods. We set up the three PBRT assays at CBER in order to compare their sensitivities, specificities, and reproducibilities. Modifications were made to the assays to eliminate one problem with the assays, viz., their high background signals. We also modified the assay such that RT activities of cellular DNA polymerases were substrantially reduced. Recently, we have adapted the PBRT assay for use with the real time quantitative system, the TaqMan system, for use with the Perkin-Elmer 7700 system. This modified assay, the TM-PERT assay, is linear over at least 6 orders magnitude and is as sensitive as the original assays. With this sensitive TM-PERT assay, we have undertaken collaborative studies with several groups at CBER to apply the assay to several regulatory issues. In a study with Kurt Brorson and colleagues (OTRR/DMA/LMDI), we are assessing whether the TM-PERT assay can be used to monitor viral clearance during the preparation of monoclonal antibodies. In a separate study with Carolyn Wilson, we determined the RT level in porcine factor VIII, a therapeutic used in those hemophiliacs when human factor VIII is unsuitable. Because the product is pig derived and because all pig cells have and may express an endogenous retrovirus (PERV), the potential exists that such factor VIII preparations have PERV. All factor VIII lots tested had low levels of PERV as assessed by the TM-PERT assay by us and by RT-PCR by Dr Wilson. Dr Wilson showed that these preparations do not contain infectious virus. As part of our investigation into the chick RT activity, we began a study to determine whether pseudotypes can form between a retrovirus core and an envelope glycoprotein (Env) from paramyxoviruses or orthomyxoviruses. If so, then this may provide a means by which the chick RT particle could enter human cells. As a model system, we investigated measles, mumps and influenza virus Envs with HIV core particles. Our results show that pseudotypes can form in vitro and thus there is a theoretical possibility of such pseudotypes forming in vivo. The consequences of such pseudotype formation remains unknown.

该项目的目标是应用和开发分子生物学方法，确保疫苗和细胞基质不受病毒，特别是逆转录病毒污染。 已经发布了三种检测逆转录酶 (RT) 的灵敏方法，其灵敏度至少比传统 RT 检测高一百万倍。 尽管这些检测方法被不同地称为 PERT（产物增强 RT）或 Amp-RT，但我们选择使用基于 PCR 的 RT 的通用名称 PBRT 来称呼它们。 这三种检测方法基于相同的原理：在 RT 反应中使用已知序列的 RNA 作为模板，并使用寡脱氧核苷酸引物；由于RNA的序列已知，因此可以通过聚合酶链式反应(PCR)扩增cDNA产物； PCR产物可以通过多种方法检测。 We set up the three PBRT assays at CBER in order to compare their sensitivities, specificities, and reproducibilities. 对测定进行了修改，以消除测定中的一个问题，即它们的高背景信号。 我们还修改了检测方法，使细胞 DNA 聚合酶的 RT 活性大大降低。最近，我们对 PBRT 测定进行了改造，使其与实时定量系统 TaqMan 系统一起使用，以便与 Perkin-Elmer 7700 系统一起使用。 这种改进的测定法（TM-PERT 测定法）在至少 6 个数量级上呈线性，并且与原始测定法一样灵敏。借助这种灵敏的 TM-PERT 检测，我们与 CBER 的多个小组进行了合作研究，将该检测应用于多个监管问题。 在与 Kurt Brorson 及其同事 (OTRR/DMA/LMDI) 的一项研究中，我们正在评估 TM-PERT 测定是否可用于监测单克隆抗体制备过程中的病毒清除情况。 In a separate study with Carolyn Wilson, we determined the RT level in porcine factor VIII, a therapeutic used in those hemophiliacs when human factor VIII is unsuitable. 由于该产品源自猪，并且所有猪细胞都具有并可能表达内源性逆转录病毒 (PERV)，因此此类 VIII 因子制剂有可能含有 PERV。 根据我们的 TM-PERT 测定和 Wilson 博士的 RT-PCR 评估，所有测试的因子 VIII 批次均具有低水平的 PERV。 威尔逊博士表明，这些制剂不含传染性病毒。 作为我们对小鸡 RT 活性研究的一部分，我们开始了一项研究，以确定副粘病毒或正粘病毒的逆转录病毒核心和包膜糖蛋白 (Env) 之间是否可以形成假型。 如果是这样，那么这可能提供了一种让小鸡 RT 颗粒进入人体细胞的方法。 As a model system, we investigated measles, mumps and influenza virus Envs with HIV core particles. 我们的结果表明假型可以在体外形成，因此理论上有可能在体内形成这种假型。 这种假型形成的后果仍然未知。