USE OF ACCESSORY GENE MUTANTS FOR THE DEVELOPMENT OF ATTENUATED HIV VACCINES

使用辅助基因突变体开发减毒 HIV 疫苗

• 批准号: 6101182
• 负责人: K PEDEN
• 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/6101182
• 关键词: AIDS vaccines; attenuated microorganism; early diagnosis; gene mutation; helper T lymphocyte; host organism interaction; human immunodeficiency virus 1; human immunodeficiency virus 2; live vaccine; macrophage; monocyte; mutant; protein structure function; tissue /cell culture; transposon /insertion element; virus protein; virus replication

The long-term goals of this project are: 1) to evaluate the feasibility of generating live attenuated virus vaccines of HIV-1 and HIV-2 that are rendered non-pathogenic by mutation of accessory genes, either individually or in combination; 2) to explore the possibility that the accessory gene proteins can be targets for anti-viral therapy. and 3) to generate retroviruses that replicate without integrating into the host genome. As prerequisites to the development of candidate live attenuated virus vaccines and the development of anti-HIV drugs directed against the accessory gene products, we have been engaged on studies to determine the role of these proteins in the life cycle of HIV-1 and HIV-2 in vitro, since a knowledge of how they function is critical to both goals. Our earlier work had demonstrated the critical role of HIV-1 Vif to virus replication in primary T cells (peripheral blood mononuclear cells, PBMC) and in primary monocyte-derived macrophages (MDM). In the case of Nef, we have shown that whether or not Nef has a measurable effect on virus replication depends on the particular virus-host system used. While Nef mutants of several HIV-1 strains all replicate slightly less well than wild type in PBMC and in MDM, there can be either no effect or dramatic reductions in virus replication when Nef mutants of HIV-1 and HIV-2 are assayed in CD4-positive cell lines. As part of our goal to develop attenuated HIV vaccine candidates, we have modified an HIV-1 genome to allow the insertion of different genes into the nef open reading frame. The vif genes of HIV-1 and HIV-2 have been inserted into the both the homologous and heterologous viruses with the aim of determining whether ectopic expression of Vif is functional and whether Vif of HIV-1 can complement Vif mutants of HIV-2 and vice versa. Preliminary work has demonstrated that HIV-2 vif can complement HIV-1 Vif mutants, but that vif from SIVagm cannot. This work will be extended to a study of Nef and Vpr. To explore the feasibility of developing a retrovirus that is replication competent but does not integrate as an obligatory step in its life cycle, we have constructed a murine leukemia virus (MLV) derivative that has an amphotropic env gene as well as mutations in the integrase gene and the terminal repeats; these latter mutations render the virus integration defective. In addition, a replication origin,ori, from the DNA virus SV40 was inserted into the viral genome. The resulting virus, MLVori, was used to infect COS7 cells, which contain the SV40 replication protein T antigen. Virus replication was observed in COS7 cells but not in CV1 cells, which do not contain SV40 T antigen. Although the virus replicated at the initial passage, multiple passages were required before a level of replication was achieved that exceeded that of the parent amphotropic MLV. The determinants of this improved replication capacity will be mapped. This approach is being extended to HIV.

本项目的长期目标是：1）评估可行性 生产HIV-1和HIV-2的减毒活病毒疫苗， 通过辅助基因突变而使其非致病性， （2）探讨是否有可能 辅助基因蛋白可以是抗病毒治疗的靶点。 和3） 来产生逆转录病毒， 基因组 作为候选减毒活疫苗开发的先决条件， 病毒疫苗和针对艾滋病毒的抗艾滋病毒药物的开发 辅助基因产物，我们一直从事研究，以确定 这些蛋白质在体外HIV-1和HIV-2生命周期中的作用， 因为了解它们如何运作对这两个目标都至关重要。 我们 早期的工作已经证明了HIV-1 Vif对病毒的关键作用， 原代T细胞（外周血单核细胞，PBMC）中的复制 和在原发性单核细胞衍生的巨噬细胞（MDM）中。 在Nef的案例中， 我们已经证明Nef是否对病毒有可测量的影响， 复制取决于所使用的特定病毒宿主系统。 当Nef 几种HIV-1病毒株的突变体的复制能力都略低于 在PBMC和MDM中的野生型，可能没有影响或显著影响 当HIV-1和HIV-2的Nef突变体 在CD 4阳性细胞系中测定。 作为我们开发减毒HIV候选疫苗目标的一部分，我们 修改了HIV-1基因组，允许将不同的基因插入 NEF开放式阅读框架。 HIV-1和HIV-2的vif基因已经被 插入到同源和异源病毒中， 目的是确定Vif的异位表达是否是功能性的， HIV-1的Vif是否可以与HIV-2的Vif突变体互补，反之亦然。 初步工作表明，HIV-2 vif可以补充HIV-1 Vif 突变体，但来自SIVagm的vif不能。 这项工作将扩大到 Nef和Vpr的研究。 探讨研制一种复制型逆转录病毒的可行性 有能力，但没有融入其生命周期的一个强制性步骤， 我们构建了一种鼠白血病病毒（MLV）衍生物， 嗜热性env基因以及整合酶基因和 末端重复序列;这些后者的突变使病毒整合 有缺陷 此外，DNA病毒还有一个复制起点ori 将SV 40插入病毒基因组中。 由此产生的病毒，MLVori， 感染COS 7细胞，该细胞含有SV 40复制蛋白 T抗原。 在COS 7细胞中观察到病毒复制，但在CV1中未观察到病毒复制 细胞，其不含SV 40 T抗原。 虽然病毒 在初始传代时复制，在传代前需要多次传代 实现了超过父级的复制级别 嗜热MLV。 这种提高复制能力的决定因素 将被映射。 这一做法正在推广到艾滋病毒。