Molecular biological methods and vaccine safety

分子生物学方法和疫苗安全性

• 批准号: 6545144
• 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/6545144
• 关键词: RNA directed DNA polymerase; Retroviridae; bioassay; biological products; chemical structure function; coagulation factor VIII; drug quality /standard; drug screening /evaluation; enzyme activity; host organism interaction; human immunodeficiency virus 1; immunologic substance development /preparation; molecular biology; nucleic acid quantitation /detection; polymerase chain reaction; public health; technology /technique development; time resolved data; tissue /cell culture; virus infection mechanism; virus protein

Summary: 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. Reverse transcriptase (RT) assays measure the presence of retroviruses. The conventional RT assay was insenstive and not quantitative. This changed with the advent of PCR-based RT PBRT) assays, which are a million-fold more sensitive than conventional RT assays. 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 two 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. We have demonstrated that mixed pseudotypes [i.e., pseudotypes containing envelope glycoproteins (Envs) from two different viruses] can form between measles virus and HIV-1 Envs. This raises the possibility that such virions can form in vivo. This may have implications for HIV pathogenesis, since HIV particles containing an Env of measles virus would be able to infect non-CD4 cells; such infected cells would have unknown consequences on HIV pathogenesis. We have initiated studies to develop quantitative assays to assess the biological activity of residual cell-substrate DNA. For many years, DNA resulting from cell substrates has been considered to pose a risk to vaccine recipients receiving products manufactured in neoplastic cells. This is one of the main reasons for these cells not being used for vaccine production to date. The risk is perceived to be either from an oncogenic potential and an infectious potential. We are developing quantitaive assays to assess both types of risks. We are developing Q-PCR assays to detect the genomes of primate polyomaviruses (SV40, JCV, BKV). Developing assays for these viruses will be a first step in our program to establish quantitative assays for the detection of adventitious agents.

摘要：该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。逆转录酶（RT）检测检测逆转录病毒的存在。传统的RT法不敏感，不能定量。随着基于pcr的RT （PBRT）检测的出现，这种情况发生了变化，其灵敏度比传统RT检测高100万倍。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。这种改进的分析，TM-PERT分析，在至少6个数量级上是线性的，并且与原始分析一样敏感。有了这种敏感的TM-PERT分析，我们已经与CBER的两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。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用于治疗血友病患者。由于该产品来源于猪，并且所有猪细胞都含有并可能表达一种内源性逆转录病毒（PERV），因此存在这种因子VIII制剂含有PERV的可能性。通过我们的TM-PERT分析和Wilson博士的RT-PCR评估，所有测试的因子VIII批次都有低水平的PERV。威尔逊博士证明这些制剂不含传染性病毒。作为我们对鸡RT活性调查的一部分，我们开始了一项研究，以确定伪型是否可以在副粘病毒或正粘病毒的逆转录病毒核心和包膜糖蛋白（Env）之间形成。如果是这样，那么这可能为小鸡RT粒子进入人类细胞提供了一种途径。作为一个模型系统，我们研究了带有HIV核心颗粒的麻疹、腮腺炎和流感病毒Envs。我们的研究结果表明，假型可以在体外形成，因此理论上存在这种假型在体内形成的可能性。这种伪型形成的后果尚不清楚。我们已经证明，麻疹病毒和HIV-1 Envs之间可以形成混合伪病毒（即含有来自两种不同病毒的包膜糖蛋白（Envs）的伪病毒）。这提高了这种病毒粒子在体内形成的可能性。这可能对艾滋病毒的发病机制有影响，因为含有麻疹病毒Env的艾滋病毒颗粒能够感染非cd4细胞；这些被感染的细胞对HIV的发病机制有未知的影响。我们已经开始研究开发定量分析来评估残留细胞底物DNA的生物活性。多年来，人们一直认为细胞底物产生的DNA对接受肿瘤细胞制造的产品的疫苗接受者构成风险。这是迄今为止这些细胞未用于疫苗生产的主要原因之一。这种风险被认为要么来自致癌潜力，要么来自感染潜力。我们正在开发定量分析来评估这两种风险。我们正在开发Q-PCR检测灵长类多瘤病毒（SV40， JCV， BKV）的基因组。开发这些病毒的检测方法将是我们建立检测外来因子的定量分析方法的第一步。