Retroviruses and safety of biologics

逆转录病毒和生物制品的安全性

• 批准号: 6839051
• 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/6839051
• 关键词: AIDS vaccines; HIV infections; Macaca nemestrina; Retroviridae; Retroviridae disease; biohazard detection; biological products; cell line; disease /disorder model; gene induction /repression; genetic strain; human immunodeficiency virus 1; latent virus infection; model design /development; polymerase chain reaction; virus genetics; virus load; virus replication

Dr. Khan's laboratory is involved in understanding the mechanisms of retrovirus latency and activation with focus towards development of strategies for early detection of endogenous retroviruses in cell substrates used in biologics. Additionally, relevant animal models are being developed for pre-clinical product evaluation and to perform prospective risk assessment of retroviruses that are of potential concern in biological products. Current projects include: 1)evaluation of various chemical and biological agents for retrovirus induction from primate vaccine cell substrates and development of sensitive assays for retrovirus detection; 2)studies of latency, replication and transmission of naturally-occurring simian foamy retrovirus isolates; and 3) development of a pre-clinical animal model for AIDS vaccine safety and efficacy. Development of a monkey model for testing antiviral agents against HIV-1. A good animal model for HIV-1 is essential for AIDS vaccine studies and development of efficacious anti-viral agents. Additionally, such a model will provide insight into the mechanism of correlates of virus infection and protection. We have obtained reproducible, long-term HIV-1 infection in pig-tailed macaques and have evaluated virus infection in animals that received high and low doses of HIV-1/LAI. Although, each animal responded uniquely to the infection, in all cases HIV-1 antibodies developed and viral sequences were detected in the PBMCs. No significant clinical symptoms were seen. The results of our study indicate that pig-tailed macaques may be a useful animal model for evaluating the efficacy of novel vaccine strategies. To investigate whether pig-tailed macaques are a good animal model for vaccine studies, we tested the efficacy of the DNA prime with protein boost strategy using our HIV-1 DNA vaccines with subsequent Env protein boost against challenge with homologous virus (HIV-1/LAI). Virus could not be detected in the plasma of animals immunized with a vaccine DNA containing the CMV promoter. PBMC DNA was analyzed by PCR using pol and gag primers to further evaluate virus infection. The results indicate that pig-tailed macaques can be used to demonstrate the efficacy of immunization strategies using HIV/LAI based vaccines against homologous challenge virus. Development and standardization of retrovirus detection assays and safety studies related to cell substrates and biological products. Vaccines and therapeutics can potentially be contaminated with infectious retrovirus due to induction of an endogenous, latent virus in the cell substrate or by recombination between endogenous retroviral sequences to produce a novel virus. Additionally, blood and blood products may contain viruses due to their presence in the host donor. Thus, to assure public health safety in biologics, it is critical to develop sensitive retrovirus detection assays and to evaluate potential risk of retrovirus infection in humans using animal models. 1) Simian foamy retrovirus (SFV) is a safety concern in biological products using simian cells and potentially in blood donors that have been exposed to non-human primates. To evaluate potential risk of SFV infection in humans, naturally-occurring SFVs were isolated from rhesus and pig-tailed macaques. A. In vitro infectivity studies indicated that the naturally-occurring macaque isolates are diverse in replication efficiency and have slower replication kinetics than laboratory-adapted prototype viruses. Current investigations are ongoing to evaluate promoter strength and transactivating factor (tas) gene function. B. Studies are underway to evaluate SFV infection in naive macaques: a)by direct injection, and b) by blood transfer. TaqMan PCR assays were developed to quantify SFV load. The animals are currently being monitored for virus infection and clinical changes. These results will provide information regarding human risk of SFV infection from macaques and address potential safety concerns regarding SFV transmission by blood donors. 2) Endogenous retroviruses can potentially contaminate biological products by activation of viral sequences in the cell susbtrate. To develop a strategy for early cell susbtrate screening for endogenous viruses, we have investigated retrovirus induction by chemicals in a well-characterized mouse cell line using a reverse transcriptase assays for detection of induced retrovirus. The results indicated inducer-dependent endogenous retrovirus activation with increased cellular DNA polymerase production. Modification of the RT assay enhanced retrovirus-specific RT detection. Vaccine cell susbtrates will be evaluated for endogenous retrovirus induction using a highly sensitive PCR based assay (PERT). This project incorporates FY2002 projects 1Z01BK003008-10, 1Z01BK003009-10, 1Z01BK003013-07, and 1Z01BK003014-07.

Khan 博士的实验室致力于了解逆转录病毒潜伏和激活的机制，重点是制定早期检测生物制剂所用细胞基质中内源逆转录病毒的策略。此外，正在开发相关的动物模型，用于临床前产品评估，并对生物制品中潜在关注的逆转录病毒进行前瞻性风险评估。 目前的项目包括：1）评估用于从灵长类疫苗细胞底物诱导逆转录病毒的各种化学和生物制剂，并开发用于逆转录病毒检测的灵敏测定法； 2）天然猿猴泡沫逆转录病毒分离株的潜伏、复制和传播研究； 3）开发艾滋病疫苗安全性和有效性的临床前动物模型。 开发用于测试 HIV-1 抗病毒药物的猴子模型。良好的 HIV-1 动物模型对于艾滋病疫苗研究和有效抗病毒药物的开发至关重要。此外，这样的模型将提供对病毒感染和保护的相关机制的深入了解。我们在猪尾猕猴中获得了可重复的长期 HIV-1 感染，并评估了接受高剂量和低剂量 HIV-1/LAI 的动物的病毒感染情况。尽管每只动物对感染的反应各不相同，但在所有情况下，HIV-1 抗体均产生，并且在 PBMC 中检测到病毒序列。未见明显临床症状。我们的研究结果表明，猪尾猕猴可能是评估新型疫苗策略功效的有用动物模型。 为了调查猪尾猕猴是否是疫苗研究的良好动物模型，我们使用 HIV-1 DNA 疫苗和随后的 Env 蛋白加强来对抗同源病毒 (HIV-1/LAI) 的攻击，测试了 DNA 初免和蛋白加强策略的功效。用含有 CMV 启动子的疫苗 DNA 免疫的动物血浆中无法检测到病毒。使用 pol 和 gag 引物通过 PCR 分析 PBMC DNA，以进一步评估病毒感染。结果表明，猪尾猕猴可用于证明使用基于 HIV/LAI 的疫苗针对同源攻击病毒的免疫策略的有效性。 与细胞底物和生物制品相关的逆转录病毒检测分析和安全性研究的开发和标准化。由于细胞基质中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，疫苗和治疗药物可能会被感染性逆转录病毒污染。此外，血液和血液制品可能含有病毒，因为它们存在于宿主供体体内。因此，为了确保生物制剂的公共卫生安全，开发灵敏的逆转录病毒检测方法并使用动物模型评估人类逆转录病毒感染的潜在风险至关重要。 1) 猿猴泡沫逆转录病毒 (SFV) 是使用猿猴细胞的生物制品以及可能接触过非人类灵长类动物的献血者的安全问题。 为了评估人类感染 SFV 的潜在风险，从恒河猴和猪尾猕猴中分离出天然存在的 SFV。 A. 体外感染性研究表明，自然产生的猕猴分离株的复制效率不同，并且复制动力学比实验室适应的原型病毒慢。目前正在进行评估启动子强度和反式激活因子 (tas) 基因功能的研究。 B. 正在进行研究评估幼猴中的 SFV 感染：a) 通过直接注射，b) 通过血液转移。 TaqMan PCR 检测是为了量化 SFV 负荷而开发的。目前正在监测这些动物的病毒感染和临床变化。这些结果将提供有关猕猴感染 SFV 的人类风险的信息，并解决有关献血者传播 SFV 的潜在安全问题。 2) 内源性逆转录病毒可能通过激活细胞基质中的病毒序列而污染生物制品。为了制定内源病毒的早期细胞底物筛选策略，我们使用逆转录酶测定法研究了化学物质在充分表征的小鼠细胞系中诱导逆转录病毒的情况，以检测诱导的逆转录病毒。结果表明，诱导剂依赖性内源性逆转录病毒激活会增加细胞 DNA 聚合酶的产生。 RT 测定的改进增强了逆转录病毒特异性 RT 检测。将使用基于高灵敏度 PCR 的测定 (PERT) 评估疫苗细胞基质的内源性逆转录病毒诱导。 该项目包括2002财年项目1Z01BK003008-10、1Z01BK003009-10、1Z01BK003013-07和1Z01BK003014-07。