Evaluation of the use of accessory gene mutants for the development of attenuat

使用辅助基因突变体开发减毒剂的评估

• 批准号: 6433511
• 负责人: KEITH 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/6433511
• 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.

该项目的长期目标是：1)评估产生HIV-1和HIV-2的减毒活病毒疫苗的可行性，这些疫苗通过单独或联合突变辅助基因而变得非致病；2)探索辅助基因蛋白可以作为抗病毒治疗的靶标的可能性；以及3)产生不整合到宿主基因组中进行复制的逆转录病毒。作为开发候选减毒活病毒疫苗和针对辅助基因产物的抗HIV药物的先决条件，我们一直在进行研究，以确定这些蛋白质在HIV-1和HIV-2体外生命周期中的作用，因为了解它们的功能对这两个目标都是至关重要的。我们早期的工作已经证明了HIV-1 Vif在原代T细胞(外周血单核细胞)和原代单核细胞来源的巨噬细胞(MDM)中对病毒复制的关键作用。在Nef的情况下，我们已经证明了Nef是否对病毒复制有可测量的影响取决于所使用的特定病毒宿主系统。虽然几个HIV-1毒株的Nef突变株在PBMC和MDM中的复制能力都略低于野生型，但当在CD4阳性细胞系中检测HIV-1和HIV-2的Nef突变株时，病毒复制要么没有影响，要么显著减少。作为我们开发减毒HIV候选疫苗的目标的一部分，我们已经修改了HIV-1基因组，允许将不同的基因插入到nef开放阅读框架中。将HIV-1和HIV-2的vif基因插入到同源和异源病毒中，目的是确定Vif的异位表达是否具有功能，以及HIV-1的Vif能否补充HIV-2的Vif突变体，反之亦然。初步研究表明，HIV-2vif可以补充HIV-1Vif突变体，但来自SIVagm的vif不能。这项工作将扩展到对Nef和VPR的研究。为了探索开发一种具有复制能力但不作为其生命周期必需步骤的逆转录病毒的可行性，我们构建了一种小鼠白血病病毒(MLV)衍生物，它具有两性包膜基因以及整合酶基因和末端重复序列的突变；这些突变导致病毒整合缺陷。此外，DNA病毒SV40的复制起点ORI被插入到病毒基因组中。由此产生的病毒MLVori被用来感染含有SV40复制蛋白T抗原的COS7细胞。在COS7细胞中观察到病毒复制，而在不含SV40T抗原的CV1细胞中未观察到病毒复制。虽然病毒在最初传代时复制，但需要多次传代才能达到超过亲本两性MLV的复制水平。将绘制这种提高的复制能力的决定因素图。这一方法正在扩展到艾滋病毒领域。