Effects Of Novel Modification Of Scd4 On Hiv1 Entry

在HIV生命周期的各个阶段中，病毒进入是设计新的治疗药物的一个有吸引力的目标。本项目的主要目的是设计和表征一种有效的进入抑制剂。此外，我们还试图确定有效抑制HIV-1进入原发CD4+ t细胞所需的基本生化要求。这些信息在设计进入抑制剂时通常是有用的。可溶性(s) CD4是临床测试的第一个进入抑制剂，失败的主要原因是它对HIV-1原代分离株的中和活性较差。此外，sCD4在低浓度下表现出增强病毒复制的内在能力。为了克服与单体sCD4相关的缺陷，我们将CD4的包膜结合域（D1D2）融合到ig - α -tp上，创建了一个重组蛋白，我们称之为D1D2- ig - α -tp。该重组蛋白质量大于650千道尔顿（kDa），具有12个gp120结合位点。12个紧密间隔的gp120结合位点的出现可以提高sCD4与膜CD4竞争病毒粒子相关gp120的能力。此外，这种蛋白的极大尺寸应该阻止激活的病毒粒子尖刺与靶细胞膜上的CCR5（大多数原发性HIV感染的重要共受体）之间的任何相互作用。我们已经在体外表达和表征了这种蛋白，并发现它对原代HIV-1分离株的包膜具有非常高的亲和力。重要的是，它能有效抑制HIV-1阳性患者外周血单核细胞的病毒复制。在病毒中和试验中，d1d2 - ig - α -tp抑制病毒复制的水平与最有效的中和抗体相当。此外，与单体sCD4不同，在次优浓度下，该重组蛋白没有进入增强活性。每一项活动都表明，我们在实现主要目标方面取得了重大进展。基于这些观察，我们正在评估d1d2 - ig - α -tp作为一种潜在的治疗剂和潜在的佐剂疫苗策略。

Among the stages of the HIV life cycle, viral entry is an attractive target for the design of new therapeutic agents. The principle purpose of this project is to design and characterize an efficient entry inhibitor. Additionally we have attempted to define the basic biochemical requirements necessary for efficient inhibition of HIV-1 entry into primary CD4+ T-cells. Such information should be generally useful in the design of entry inhibitors. Soluble (s) CD4, the first entry inhibitor to be tested clinically, failed primarily because it demonstrated poor neutralizing activity against primary isolates of HIV-1. In addition, sCD4 manifested an intrinsic capacity to enhance viral replication at low concentrations. To overcome the deficiencies associated with monomeric sCD4 we fused the envelope binding domain of CD4 (D1D2) to Ig-alpha-tp creating a recombinant protein we term D1D2-Ig-alpha-tp . This recombinant protein exhibited a mass greater than 650 kilodaltons (kDa) and present twelve gp120 binding sites. The presentation of twelve closely spaced gp120 binding sites should improve the capacity of sCD4 to compete with membrane CD4 for virion associated gp120. In addition, the extremely large size of this protein should preclude any interaction between activated virion spikes and CCR5, the important co-receptor for most primary HIV infections, on the target cell membrane. We have expressed and characterized this protein in vitro and have found that it exhibits an extraordinarily high affinity for the envelopes of primary HIV-1 isolates. Importantly it efficiently inhibits the replication of virus from HIV-1 positive patient PBMCs. In viral neutralization assays D1D2-Ig-alpha-tp inhibits viral replication at levels comparable to the most potent neutralizing antibodies available. Moreover, unlike monomeric sCD4, at suboptimal concentrations this recombinant protein show no entry-enhancing activity. Each of these activities suggest that we have progressed significantly in our principle goals. Based on these observations we are evaluating D1D2-Ig-alpha-tp as a potential therapeutic agent and a potential adjuvant vaccine strategy.