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

该项目的目标是应用和开发分子生物学方法，以确保疫苗和细胞基质不受病毒，特别是逆转录病毒污染。 已经发表了三种灵敏的检测逆转录酶（RT）的方法，其灵敏度比常规RT测定法高至少一百万倍。 尽管这些试验被称为PERT，（对于产物增强的RT）或Amp-RT，对于基于PCR的RT，我们选择用PBRT的通用名称来称呼它们。这三种测定依赖于相同的原理：在RT反应中，已知序列的RNA用作具有寡脱氧核苷酸引物的模板;因为RNA的序列是已知的，所以cDNA产物可以通过聚合酶链式反应（PCR）扩增; PCR产物可以通过许多方法检测。 我们在CBER建立了三种PBRT检测方法，以比较它们的灵敏度、特异性和重现性。 对测定法进行了修改以消除测定法的一个问题，即，高背景信号。 我们还修改了测定法，使RT细胞DNA聚合酶的活动大幅减少。 最近，我们已将PBRT测定法与真实的时间定量系统TaqMan系统一起使用，以与Perkin-Elmer 7700系统一起使用。 这种改良的PBRT测定，即TM-PERT测定，在至少6个数量级上呈线性，并且与原始PBRT测定一样灵敏。 有了这种敏感的TM-PERT检测，我们已经与CBER的几个小组进行了合作研究，将该检测应用于几个监管问题。 在Kurt Brorson及其同事的一项研究（OTRR/DMA/LMDI）中，我们正在评估TM-PERT测定法是否可用于监测单克隆抗体制备过程中的病毒清除。目前，工业上使用的方法有电子显微镜和感染性测定。 这些分析是昂贵的，耗时的，非定量的，并且变化很大。 应用敏感的和定量的TM-PERT测定来跟踪病毒清除具有明显的吸引力。 初步实验表明，该测定法将适用于该功能。 在与Carolyn Wilson进行的一项单独研究中，我们测定了猪因子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。 由于产品来源于猪细胞，并且由于所有猪细胞均具有且部分细胞可能表达内源性逆转录病毒（PERV），因此猪因子VIII制剂可能具有PERV。 根据我们通过TM-PERT试验和Carolyn Wilson通过RT-PCR进行的评估，检测的所有因子VIII批次均具有低水平的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道理的。

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.