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

该项目的长期目标是：1）评估可行性 生产 HIV-1 和 HIV-2 减毒活病毒疫苗 通过辅助基因突变而变得非致病性，或者 单独或组合； 2）探索可能性 辅助基因蛋白可以成为抗病毒治疗的靶标。 和 3) 生成无需整合到宿主中即可复制的逆转录病毒 基因组。 作为候选活体减毒发展的先决条件 病毒疫苗和针对艾滋病病毒的抗艾滋病药物的开发 辅助基因产品，我们一直致力于研究以确定 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于实现这两个目标都至关重要。 我们的 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 以及原代单核细胞衍生的巨噬细胞（MDM）。 就内夫而言， 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。 而内夫 几种 HIV-1 病毒株的突变体的复制效果均略低于 野生型在 PBMC 和 MDM 中，可能没有效果或显着 当 HIV-1 和 HIV-2 的 Nef 突变体被抑制时，病毒复制减少 在 CD4 阳性细胞系中进行测定。 作为我们开发减毒 HIV 候选疫苗目标的一部分，我们已经 修改了 HIV-1 基因组以允许将不同的基因插入 nef开放阅读框。 HIV-1 和 HIV-2 的 vif 基因已被 插入同源和异源病毒中 确定 Vif 异位表达是否具有功能性和 HIV-1 的 Vif 是否可以补充 HIV-2 的 Vif 突变体，反之亦然。 初步工作表明 HIV-2 vif 可以补充 HIV-1 Vif 突变体，但 SIVagm 的 vif 不能。 这项工作将延伸至 Nef 和 Vpr 的研究。 探索开发可复制逆转录病毒的可行性 有能力，但不作为其生命周期中的强制性步骤进行整合， 我们构建了一种鼠白血病病毒（MLV）衍生物，它具有 两性 env 基因以及整合酶基因和 末端重复；后面这些突变使病毒整合 有缺陷。 此外，复制起点，ori，来自DNA病毒 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，但在 CV1 细胞中未观察到病毒复制 细胞，不含 SV40 T 抗原。 虽然病毒 在最初的传代中复制，之前需要多次传代 复制水平超过了父代 双嗜性 MLV。 复制能力提高的决定因素 将被映射。 这种方法正在扩展到艾滋病毒。

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.