Accessory gene mutants for attenuated HIV vaccines

总结： 本项目的长期目标是：1）评估通过单独或联合突变辅助基因来产生非致病性HIV-1和HIV-2减毒活病毒疫苗的可行性; 2）探索辅助基因蛋白作为抗病毒治疗靶点的可能性;和3）产生复制而不整合到宿主基因组中的逆转录病毒。 作为开发候选减毒活病毒疫苗和开发针对辅助基因产物的抗艾滋病毒药物的先决条件，我们一直在进行研究，以确定这些蛋白质在体外HIV-1和HIV-2生命周期中的作用，因为了解它们如何发挥作用对这两个目标至关重要。 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，PBMC）和原代单核细胞衍生的巨噬细胞（MDM）中对病毒复制的关键作用。 在Nef的情况下，我们已经表明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。 虽然几种HIV-1毒株的Nef突变体在PBMC和MDM中的复制都略低于野生型，但当在CD 4阳性细胞系中测定HIV-1和HIV-2的Nef突变体时，病毒复制可能没有影响或显著减少。 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，允许将不同的基因插入nef开放阅读框架。 将HIV-1和HIV-2的vif基因插入同源和异源病毒中，目的是确定Vif的异位表达是否有功能，以及HIV-1的Vif是否可以补充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突变体，但SIVagm的vif不能。 这项工作将扩展到Nef和Vpr的研究。 该小组最近对HIV-1的vpu基因产生了兴趣，因为已经发现HIV-1的几个克隆具有突变的vpu基因，并且已经证明Vpu可以增强MDM的感染性。 例如，AD和MAL克隆都具有导致Vpu-负病毒的突变。 AD中vpu突变的纠正增强了其在MDM中的复制。 MAL是不能在MDM中建立生产性感染的R5病毒的少数实例之一，尽管MAL Env可以介导进入MDM和MDDC。该小组正试图绘制巨噬细胞嗜性的决定因素，并正在使用MAL。确定是否Vpu-plus版本的MAL现在能够在MDM中复制是其中一个步骤。 如果这还不够，那么将在M嗜性病毒和MAL之间构建杂交病毒。

Summary: 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. The group has recently become interested in the vpu gene of HIV-1 because of the fact that several clones of HIV-1 have been found to have a mutated vpu gene and Vpu has been shown to enhance infectivity in MDM. For example, both the AD and the MAL clones had mutations that resulted in a Vpu-minus virus. Correction of the vpu mutation in AD enhanced its replication in MDM. MAL is one of the few examples of an R5 virus that cannot establish a productive infection in MDM despite the fact that the MAL Env can mediate entry into MDM and MDDC. The group is trying to map the determinants of macrophage tropism and are using MAL. Determining whether a Vpu-plus version of MAL is now able to replicate in MDM is one of the steps. If this is not sufficient, then hybrid viruses will be constructed between an M-tropic virus and MAL.