Retroviruses and safety of biologics

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，重点是开发用于生物制品中细胞基质中内源性逆转录病毒的早期检测策略。此外，正在开发用于临床前产品评价的相关动物模型，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）评价用于从灵长类疫苗细胞基质诱导逆转录病毒的各种化学和生物试剂，并开发用于逆转录病毒检测的灵敏测定; 2）研究天然存在的猿猴泡沫逆转录病毒分离株的潜伏期、复制和传播;和3）开发用于AIDS疫苗安全性和有效性的临床前动物模型。 用于测试抗HIV-1的抗病毒剂的猴模型的开发。良好的HIV-1动物模型对于艾滋病疫苗研究和开发有效的抗病毒药物至关重要。此外，这样的模型将提供深入了解病毒感染和保护的相关机制。我们已经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的动物中的病毒感染。虽然每只动物对感染的反应都是独特的，但在所有情况下，都产生了HIV-1抗体，并在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，猪尾猕猴可能是一个有用的动物模型，用于评估新的疫苗策略的有效性。 为了研究猪尾猕猴是否是疫苗研究的良好动物模型，我们使用我们的HIV-1 DNA疫苗测试了DNA初免与蛋白加强策略的效力，随后使用Env蛋白加强对抗同源病毒（HIV-1/LAI）的攻击。在用含有CMV启动子的疫苗DNA免疫的动物的血浆中不能检测到病毒。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针对同源攻击病毒的基于HIV/LAI的疫苗的免疫策略的功效。 逆转录病毒检测方法的开发和标准化以及与细胞基质和生物制品相关的安全性研究。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，疫苗和治疗剂可能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中。因此，为了确保生物制品的公共卫生安全，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)猴泡沫逆转录病毒（SFV）是使用猴细胞的生物制品中的安全性问题，可能存在于暴露于非人灵长类动物的献血者中。 为了评估SFV感染人类的潜在风险，从恒河猴和猪尾猕猴中分离天然存在的SFV。 A.体外感染性研究表明，自然发生的猕猴分离株的复制效率不同，复制动力学比实验室适应的原型病毒慢。目前的调查正在进行中，以评估启动子强度和反式激活因子（tas）基因的功能。 B。正在进行研究以评估SFV在未感染猕猴中的感染：a）通过直接注射，和B）通过血液转移。开发了TaqMan PCR测定以定量SFV负载。目前正在监测动物的病毒感染和临床变化。这些结果将提供关于人类从猕猴感染SFV风险的信息，并解决关于献血者传播SFV的潜在安全性问题。 2)内源性逆转录病毒可通过激活细胞基质中的病毒序列而潜在地污染生物制品。为了开发内源性病毒的早期细胞底物筛选策略，我们使用逆转录酶测定法检测诱导的逆转录病毒，研究了化学品在充分表征的小鼠细胞系中的逆转录病毒诱导。结果表明，诱导剂依赖的内源性逆转录病毒激活与细胞DNA聚合酶的生产增加。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的试验（PERT）评价疫苗细胞悬液的内源性逆转录病毒诱导。 本项目包含2002财年项目1 Z 01 BK 003008 -10、1 Z 01 BK 003009 -10、1 Z 01 BK 003013 -07和1 Z 01 BK 003014 -07。

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.