DEVELOPMENT AND STANDARDIZATION OF RETROVIRUS DETECTION ASSAYS AND SAFETY STUDIES

逆转录病毒检测分析和安全性研究的开发和标准化

• 批准号: 6293730
• 负责人: Arifa S Khan
• 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/6293730
• 关键词: RNA directed DNA polymerase; Retroviridae; animal tissue; diagnosis quality /standard; drug quality /standard; human immunodeficiency virus 1; method development; microorganism culture; polymerase chain reaction; simian immunodeficiency virus; tissue /cell culture; viral vaccines; virus replication

Biological products can potentially be contaminated with an infectious retrovirus, which can arise due to induction of an endogenous, latent virus or by recombination between endogenous retroviral sequences to produce a novel virus. Thus sensitive retrovirus detection assays are necessary to demonstrate the absence of known and novel retroviral contaminants in cell substrates used for production of vaccines and therapeutics and for screening xenotransplants. 1) Reverse transcriptase activity has been found in chicken cell-derived vaccines using the highly sensitive PERT assay. The RT activity is produced from the cell substrate and is associated with endogenous retroviral sequences related to ALV and EAV. Infectivity studies using a variety of human cell lines and PBMCs indicated the absence of replication-competent virus in the RT activity produced from chicken embryo fibroblasts. Furthermore, no replicating retrovirus was detected by the PERT assay in human PBMCs inoculated with measles vaccine material. Highly sensitive PCR assays, including Alu-PCR, which can specifically detect viral-cell integration junctions, were used to investigate entry and integration of EAV and ALV sequences in human PBMC DNA that had been inoculated with measles vaccine material. These studies indicated the absence of infectious retrovirus in the measles vaccine and alleviated public concerns regarding vaccine safety. 2) Simian foamy virus (SFV) is highly prevalent in non-human primates and can infect humans by cross-species transfer from infected animals. Sensitive biological and molecular assays were developed to identify SFV infections and to screen biological products. In an effort to evaluate potential outcome of SFV infection in humans, studies are underway to investigate virus replication of primary SFV isolates by infection of different human cell lines and by functional analysis of their LTRs in CAT assays. A biological detection assay for quantitative assessment of SFV is also being developed. These studies will provide important information regarding mechanism and determinants of virus replication which may be useful in assessing the potential risk of SFV cross-infection in humans. 3) Retrovirus induction assay using IdU has been established to investigate the presence of latent, infectious retroviruses in vaccine cell substrates. PERT analysis of IdU-induced K-Balb mouse cells indicated early and late production of retrovirus from the induced cells. Retrovirus production correlated with inducer dose. Studies are underway to analyze the induction of different retrovirus types from the mouse cells. Additional studies are directed towards optimizing induction conditions for activation of endogenous retroviruses from primate cells. These results may help in developing a general strategy for sensitive detection of latent, infectious retroviruses in vaccine cell substrates.

生物制品可能被感染性逆转录病毒污染，这可能是由于内源性潜伏病毒的诱导或内源性逆转录病毒序列之间的重组产生新病毒而引起的。因此，敏感的逆转录病毒检测试验是必要的，以证明在用于生产疫苗和治疗剂以及用于筛选异种移植物的细胞基质中不存在已知和新型逆转录病毒污染物。1)使用高灵敏度PERT测定法，在鸡细胞衍生疫苗中发现了逆转录酶活性。RT活性由细胞底物产生，并与ALV和EAV相关的内源性逆转录病毒序列相关。使用多种人细胞系和PBMC的增殖性研究表明，在从鸡胚成纤维细胞产生的RT活性中不存在有复制能力的病毒。此外，通过PERT试验在接种麻疹疫苗材料的人PBMC中未检测到复制型逆转录病毒。高灵敏度的PCR检测，包括RT-PCR，可以特异性地检测病毒-细胞整合连接，被用来调查的EAV和ALV序列的进入和整合的人PBMC DNA已接种麻疹疫苗材料。这些研究表明麻疹疫苗中不含传染性逆转录病毒，减轻了公众对疫苗安全性的担忧。 2)猴泡沫病毒（SFV）在非人灵长类动物中高度流行，并可通过受感染动物的跨物种转移感染人类。开发了灵敏的生物学和分子检测方法来鉴定SFV感染和筛选生物制品。为了评估SFV感染人类的潜在结果，正在进行研究以通过感染不同的人类细胞系并通过在CAT测定中对其LTR进行功能分析来研究原代SFV分离株的病毒复制。用于定量评估SFV的生物检测测定也正在开发中。这些研究将提供有关病毒复制机制和决定因素的重要信息，这可能有助于评估人类SFV交叉感染的潜在风险。 3)已建立使用IdU的逆转录病毒诱导试验，以研究疫苗细胞底物中是否存在潜伏的感染性逆转录病毒。IDU诱导的K-Balb小鼠细胞的PERT分析表明从诱导的细胞中产生逆转录病毒的早期和晚期。逆转录病毒产量与诱导剂剂量相关。研究正在进行中，以分析从小鼠细胞诱导不同的逆转录病毒类型。另外的研究针对优化用于从灵长类细胞激活内源性逆转录病毒的诱导条件。这些结果可能有助于开发一种敏感检测疫苗细胞基质中潜伏的传染性逆转录病毒的一般策略。