LENTIVIRUS ATTENUATION THROUGH HIGH FIDELITY REPLICATION

通过高保真复制实现慢病毒衰减

• 批准号: 6313305
• 负责人: Stephen Dewhurst
• 金额: $ 25.29万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-08 至 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6313305
• 关键词: AIDS vaccines; Macaca mulatta; attenuated microorganism; cytotoxic T lymphocyte; gene mutation; leukocyte activation /transformation; mutant; recombinant virus; simian immunodeficiency virus; tissue /cell culture; vaccine development; virus load; virus replication

Human Immunodeficiency Virus type-1 (HIV-1) reverse transcriptase (RT) is a highly error-prone enzyme that is thought to be responsible for the generation of viral genetic diversity. This in turn has been suggested to be essential to the virus' ability to evade host immune responses, and to establish a state of persistent, productive infection in the host. Our hypothesis is that viruses with enhanced replicational fidelity will generate fewer mutants than wild-type viruses, and that such "high- fidelity" viruses may be incapable of escaping from host immune pressure, due to their inability to spawn highly diverse quasispecies. If correct, this hypothesis could have important implications for the design of live-attenuated HIV vaccines. This application represents a proof-of-concept study that is designed to test whether a "high fidelity" primate lentivirus is indeed attenuated in terms of the generation of new mutants and escape from immune pressure within a nonhuman primate host. The Simian Immunodeficiency Virus (SIV)/macaque model system will be used for these experiments. In earlier studies, we have created HIV-1 and SIV RT mutants with increased replicational fidelity (approx. 10-fold greater than wild-type). Similar methods will be used to create several high-fidelity mutants of SIVmac239 RT. After biochemical characterization, these RT mutants will be substituted into an intact SIVmac239 molecular clone, and virus stocks will be generated (SIVmac239-hifi). The replicational fitness and fidelity of these viruses will be tested in vitro, and a clone with wild-type replicational fitness but enhanced replicational fidelity will then be selected for in vivo studies. This virus will be inoculated into rhesus macaques (both Mamu-A*01 positive and Mamu-A*01 negative animals), and the development of virus-specific, Mamu-A*01 restricted CTL responses will be examined using MHC:peptide tetramers. Plasma virus load will be measured at selected time points following infection, and viral genetic diversity will be analyzed at several loci, including a Mamu-A*01 restricted Tat CTL epitope that has previously been to shown to undergo very rapid replacement in SIVmac239-infected, Mamu-A*01 positive rhesus macaques, but not in infected Mamu-A*01 negative animals. These experiments are expected to provide insight as to the potential utility of using high fidelity RT mutants to improve the safety of live-attenuated HIV vaccines.

人类免疫缺陷病毒类型1（HIV-1）逆转录酶（RT）是一种高度易错的酶，被认为是导致病毒遗传多样性产生的原因。反过来，这反过来被认为是病毒逃避宿主免疫反应并在宿主中建立持续性，生产性感染状态的能力。我们的假设是，具有增强复制保真度的病毒比野生型病毒会产生更少的突变体，并且这种“高富达”病毒可能无法逃避宿主免疫压力，因为它们无法产生高度多样化的准序列。如果正确，该假设可能对生命衰减的HIV疫苗的设计具有重要意义。该应用代表了一项概念验证研究，旨在测试“高保真”灵长类动物慢病毒是否确实会因产生新突变体的产生并逃避非人类灵长类动物宿主中的免疫压力而衰减。这些实验将使用Simian免疫缺陷病毒（SIV）/猕猴模型系统。在较早的研究中，我们创建了具有增加复制保真度的HIV-1和SIV RT突变体（比野生型大约10倍）。类似的方法将用于创建SIVMAC239 RT的几个高保真突变体。生化表征后，这些RT突变体将被取代为完整的SIVMAC239分子克隆，并产生病毒量（SIVMAC239-HIFI）。这些病毒的复制适应性和忠诚度将在体外进行测试，并具有野生型复制适应性的克隆，但将选择增强的复制保真度进行体内研究。该病毒将被接种到恒河猕猴（Mamu-A*01阳性和Mamu-A*01阴性动物）中，并将使用MHC：肽四聚体进行病毒特异性，MAMU-A*01限制的CTL反应的发展。 Plasma virus load will be measured at selected time points following infection, and viral genetic diversity will be analyzed at several loci, including a Mamu-A*01 restricted Tat CTL epitope that has previously been to shown to undergo very rapid replacement in SIVmac239-infected, Mamu-A*01 positive rhesus macaques, but not in infected Mamu-A*01 negative animals.预计这些实验将提供有关使用高保真RT突变体提高活体衰减HIV疫苗安全性的潜在实用性的洞察力。