Safety issues in viral vaccines and cell substrates

该计划分为几个部分。第一个是与电池底物安全相关的问题。第二个方向是开发不定物质的定量分析方法。第三个是关于了解艾滋病毒在艾滋病毒疾病期间是如何演变的，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疗法的设计相关。 对于致瘤或来源于肿瘤的细胞底物，尚未解决的主要问题之一是残留的细胞底物DNA是否对疫苗接受者构成安全风险。这种风险可能来自DNA的致癌活性，如果DNA包含激活的显性癌基因(病毒的细胞)，或者如果DNA包含DNA病毒的基因组或逆转录病毒的前病毒拷贝，则可能来自其传染性。目前正在开发定量分析方法，以便能够评估这两种活动的风险。一种检测啮齿动物体内接种的细胞癌基因的体内检测方法正在开发中。建立了一种体外定量逆转录病毒感染性DNA的方法，结果表明，仅能检测到1pg的克隆逆转录病毒基因组和5g感染细胞的DNA。目前的工作针对的是确定降低DNA传染性的方法。 关于不定因素，我们与Andrew Lewis(DVP/OVRR/CBER)合作，开发了灵长类多瘤病毒JCV、BKV和SV40的实时定量PCR检测方法。目前，刘易斯博士和我与Ed Tabor博士(OBRR)合作，正在使用Q-PCR分析来调查最初在脊髓灰质炎病毒和腺病毒疫苗中发现的SV40是否是血液供应中的污染物，如果是，是什么水平。BKV和JCV将作为阳性对照。我们还在开发免疫学测试，以回答人类是否存在SV40抗体，以及这些抗体是否具有中和性。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。有了这些中和试验，我们将首次能够确定人血清中三种多瘤病毒的中和滴度。 我们已经开始了一项小型研究，以开发一种针对SARS冠状病毒的中和试验。这将涉及产生假型病毒，并使用从感染者身上提纯的抗体。 我们在HIV方面的工作主要集中在确定HIV使用什么辅助受体进入各种细胞，以及HIV是否能够适应使用其中一种次要的辅助受体。了解艾滋病毒如何适应以及与什么受体相适应将有助于疫苗设计和疫苗安全性。我们已经鉴定出一种使用CCR5的HIV毒株，它不能很好地利用CXCR6进入，但在传代后会适应并很好地利用它。由于这种受体在胎盘组织和胸腺中表达，因此有可能HIV在这些组织中使用CXCR6，或者HIV在HIV疾病的某些阶段适应使用这种受体。重要的是，同时表达CXCR6和CXCR4的细胞的额外传代导致了一种现在可以使用CXCR4的病毒的进化。包膜基因的变化正在确定中。 本项目包含2002财年项目1Z01BK003011-09、1Z01BK003012-09和1Z01BK003017-07。

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.