Safety issues in viral vaccines and cell substrates

病毒疫苗和细胞基质的安全问题

• 批准号: 6839053
• 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/6839053
• 关键词: Coronaviridae; DNA virus; Polyomavirus; biohazard detection; blood bank /supply contamination; drug adverse effect; drug screening /evaluation; human immunodeficiency virus; neutralizing antibody; oncogenes; oncogenic virus; polymerase chain reaction; technology /technique development; vaccine evaluation; viral vaccines; virus infection mechanism; virus receptors

The program is divided into several parts. The first is related to issues associated with the safety of cell substrates. The second is directed to developing quantitative assays for adventitious agents. The third is concerned with understanding how HIV evolves during HIV disease, what coreceptors the virus uses to enter cells, and what are the determinants of tropism; this information will be relevant to the design of AIDS vaccines and therapeutics. One of the major issues that has yet to be resolved with respect to cell substrates that are either tumorigenic or are derived from tumors is whether residual cell-substrate DNA poses a safety risk to vaccine recipients. This risk could be from the oncogenic activity of the DNA, if the DNA contains activated dominant oncogenes (cellular of viral), or from its infectivity, if the DNA contains the genome of a DNA virus or the proviral copy of a retrovirus. Quantitative assays are being developed to be able to assess the risk for both these activities. An in vivo assay to detect inoculated cellular oncogenes in rodents is being developed. An in vitro assay to quantify infectious DNA of a retrovirus has been developed and has revealed that as little as 1 pg of cloned retroviral genome and 5 ?g of DNA from infected cells can be detected. Current work is directed towards determining methods to reduce DNA infectivity. With respect to adventitious agents, in collaboration with Andrew Lewis (DVP/OVRR/CBER), we have developed quantitative, real-time PCR assays for the primate polyomaviruses JCV, BKV, and SV40. Currently, Dr Lewis and I, in collaboration with Dr Ed Tabor (OBRR), are using the Q-PCR assays to investigate whether SV40, originally discovered as a contaminant in poliovirus and adenovirus vaccines, is a contaminant in the blood supply and, if so, at what level. BKV and JCV will serve as positive controls. We are also developing immunological assays to answer whether antibodies to SV40 are present in humans and whether these antibodies are neutralizing. For this, we have developed a reporter-gene based assay for SV40 and are developing analogous assays for BKV and JCV. With these neutralization assays, we will be able for the first time to determine the neutralization titers for the three polyomaviruses in human serum. We have initiated a small study to develop a neutralization assay for the SARS coronavirus. This will involve generating pseudotyped viruses and using antibodies purified from infected individuals. Our work on HIV has centered on determining what coreceptors HIV uses to enter various cells and whether HIV can adapt to use one of the minor coreceptors. Understanding how HIV adapts and to what receptor will assist in vaccine design and vaccine safety. We have identified a CCR5-using HIV strain that can use CXCR6 poorly for entry but that adapts on passage to use it well. As this receptor is expressed in placental tissue and in the thymus, the possibility exists that HIV uses CXCR6 in these tissues or that HIV adapts to use this receptor at certain stages in HIV disease. Importantly, additional passage in cells expressing both CXCR6 and CXCR4, one of the major coreceptors, results in the evolution of a virus that now can use CXCR4. Changes in the envelope genes are being determined. This project incorporates FY2002 projects 1Z01BK003011-09, 1Z01BK003012-09, and 1Z01BK003017-07.

该程序分为几个部分。 第一个与细胞基质安全性相关的问题有关。第二个目标是开发外来因子的定量分析方法。第三个涉及了解艾滋病毒在艾滋病毒疾病期间如何进化，病毒使用哪些辅助受体进入细胞，以及趋向性的决定因素是什么；这些信息将与艾滋病疫苗和治疗方法的设计相关。 关于致瘤性或源自肿瘤的细胞基质，尚未解决的主要问题之一是残留的细胞基质 DNA 是否对疫苗接受者构成安全风险。 如果 DNA 含有激活的显性致癌基因（细胞或病毒），则这种风险可能来自 DNA 的致癌活性；如果 DNA 含有 DNA 病毒的基因组或逆转录病毒的原病毒副本，则这种风险可能来自其感染性。 定量分析正在开发中，以便能够评估这两种活动的风险。 正在开发一种检测啮齿动物中接种的细胞癌基因的体内测定法。 一种定量逆转录病毒感染性 DNA 的体外测定方法已经开发出来，并表明可以检测到少至 1 pg 的克隆逆转录病毒基因组和来自受感染细胞的 5 µg DNA。 目前的工作旨在确定降低 DNA 感染性的方法。 关于外来因子，我们与 Andrew Lewis (DVP/OVRR/CBER) 合作，开发了灵长类多瘤病毒 JCV、BKV 和 SV40 的定量实时 PCR 检测方法。 目前，Lewis 博士和我与 Ed Tabor 博士 (OBRR) 合作，正在使用 Q-PCR 检测来调查最初作为脊髓灰质炎病毒和腺病毒疫苗中的污染物发现的 SV40 是否是血液供应中的污染物，如果是，其水平如何。 BKV 和 JCV 将作为阳性对照。 我们还在开发免疫学检测方法，以确定人类体内是否存在 SV40 抗体以及这些抗体是否具有中和作用。 为此，我们开发了一种基于报告基因的 SV40 检测方法，并正在开发 BKV 和 JCV 的类似检测方法。 通过这些中和测定，我们将能够首次确定人血清中三种多瘤病毒的中和滴度。 我们已经启动了一项小型研究，开发 SARS 冠状病毒的中和测定方法。 这将涉及生成假型病毒并使用从感染个体中纯化的抗体。 我们在艾滋病毒方面的工作集中于确定艾滋病毒使用哪些辅助受体进入各种细胞，以及艾滋病毒是否能够适应使用其中一种次要辅助受体。 了解艾滋病毒如何适应以及适应什么受体将有助于疫苗设计和疫苗安全。 我们已经鉴定出一种使用 CCR5 的 HIV 毒株，它在进入时不能很好地利用 CXCR6，但在传代时会适应并很好地利用它。 由于该受体在胎盘组织和胸腺中表达，因此存在 HIV 在这些组织中使用 CXCR6 的可能性，或者 HIV 在 HIV 疾病的某些阶段适应使用该受体的可能性。 重要的是，在表达 CXCR6 和 CXCR4（主要共同受体之一）的细胞中进行额外传代，导致现在可以使用 CXCR4 的病毒进化。 正在确定包膜基因的变化。 该项目包括 2002 财年项目 1Z01BK003011-09、1Z01BK003012-09 和 1Z01BK003017-07。