APPLICATION AND DEVELOPMENT OF MOLECULAR BIOLOGICAL METHODS TO THE ISSUES OF VACC

分子生物学方法在 VACC 问题中的应用和发展

• 批准号: 6293733
• 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/6293733
• 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 PBRT assay, the TM-PERT assay, is linear over at least 6 orders magnitude and is as sensitive as the original PBRT 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. Currently, methods such electron microscopy and infectivity assays are used by industry. These assays are costly, time-consuming, non-quantitative, and quite variable. The application of the senstive and quantitative TM-PERT assay to follow viral clearance has obvious attractions. Preliminary experiments suggest that the assay will be suitable for this function. In a separate study with Carolyn Wilson, we determined the RT level in porcine factor VIII, a therapeutic used in a small percentage of hemophiliacs when human factor VIII is unsuitable. Because the product is derived from pig cells and because all pig cells have and some may express an endogenous retrovirus (PERV), the potential exists that porcine 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 Carolyn Wilson. Fortunately, Dr Wilson has shown that these factor VIII preparations do not contain infectious virus, and thus their continued use is warranted.

这个项目的目标是应用和发展分子生物学方法，以确保疫苗和细胞底物不受病毒，特别是逆转录病毒的污染。已经发表了三种检测逆转录酶（RT）的灵敏方法，它们的灵敏度至少是传统RT测定法的一百万倍。虽然这些检测方法被称为PERT（用于产品增强RT）或Amp-RT，但我们选择用PBRT的通用名称来称呼它们，用于基于pcr的RT。这三种检测方法依赖于相同的原理：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序列已知，cDNA产物可通过聚合酶链反应（PCR）扩增；PCR产物可以通过多种方法检测。我们在CBER建立了三种PBRT检测方法，以比较它们的敏感性、特异性和可重复性。对测定法进行了修改，以消除测定法的一个问题，即它们的高背景信号。我们还修改了实验，使细胞DNA聚合酶的RT活性大大降低。最近，我们已经调整了PBRT分析与实时定量系统TaqMan系统一起使用，用于Perkin-Elmer 7700系统。这种改进的PBRT检测，即TM-PERT检测，在至少6个数量级上呈线性，并且与原始的PBRT检测一样敏感。有了这种敏感的TM-PERT分析，我们已经与CBER的几个小组进行了合作研究，将该分析应用于几个监管问题。在与Kurt broson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。目前，工业上使用的方法是电子显微镜和感染性测定法。这些检测是昂贵的，耗时的，非定量的，而且变化很大。应用灵敏、定量的TM-PERT分析跟踪病毒清除具有明显的吸引力。初步实验表明，该方法适用于该功能。在与Carolyn Wilson的另一项研究中，我们确定了猪因子VIII的RT水平，当人因子VIII不适合时，猪因子VIII用于一小部分血友病患者的治疗。由于该产品来源于猪细胞，而且所有猪细胞都含有内源性逆转录病毒（PERV），有些细胞可能表达这种病毒，因此猪因子VIII制剂可能含有PERV。通过我们的TM-PERT分析和Carolyn Wilson的RT-PCR评估，所有测试的因子VIII批次都有低水平的PERV。幸运的是，威尔逊博士已经证明这些因子VIII制剂不含传染性病毒，因此它们的继续使用是有保证的。