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

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 由于辅助基因突变而变得非致病的 单独或结合在一起；2)探索 辅助基因蛋白可以成为抗病毒治疗的靶点。和3) 生成不整合到宿主中即可复制的逆转录病毒 基因组。作为候选人发展的先决条件，生活衰减 针对人类免疫缺陷病毒的病毒疫苗和抗HIV药物的开发 辅助基因产物，我们一直在进行研究，以确定 这些蛋白在HIV-1和HIV-2体外生命周期中的作用 因为对它们如何运作的了解对这两个目标都至关重要。我们的 早期的研究已经证明了HIV-1 Vif对病毒的关键作用 原代T细胞(外周血单核细胞)的复制 原代单核细胞来源的巨噬细胞(MDM)。在Nef的案例中， 我们已经证明了Nef对病毒是否有可测量的影响 复制取决于使用的特定病毒主机系统。而Nef 几种HIV-1毒株的突变体的复制能力都略低于 在PBMC和MDM中，野生型要么没有作用，要么戏剧性地 当HIV-1和HIV-2的Nef突变株 在CD4阳性细胞系中进行检测。 作为我们开发减毒HIV候选疫苗目标的一部分，我们有 修改HIV-1基因组以允许将不同的基因插入到 NEF开放阅读框架。HIV-1和HIV-2的vif基因已经被 插入到同源和异源病毒中 目的确定Vif的异位表达是否具有功能性和 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-2 VIF可以补充HIV-1 VIF 变种人，但来自SIVagm的vif不能。这项工作将扩展到 Nef和VPR的研究。 探索开发可复制的逆转录病毒的可行性 有能力，但不作为其生命周期的强制性步骤进行整合， 我们已经构建了一种小鼠白血病病毒(MLV)的衍生物，它具有 两性包膜基因以及整合酶基因和 末端重复；这些后一种突变使病毒整合 有缺陷的。此外，来自DNA病毒的复制起始点ORI SV40被插入到病毒基因组中。由此产生的病毒MLVori， 用于感染含有SV40复制蛋白的COS7细胞 T抗原。在COS7细胞中观察到病毒复制，而在CV1细胞中未观察到病毒复制 不含SV40T抗原的细胞。尽管这种病毒 在最初的通道中复制，之前需要多个通道 实现的复制级别超过了父级 两性MLV。这种复制能力提高的决定因素 将会被映射。这一方法正在扩展到艾滋病毒领域。

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.