Effects Of Novel Modification Of Scd4 On Hiv1 Entry

在艾滋病毒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。这个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进入原代CD4+T细胞所必需的基本生化要求。这些信息在设计进入抑制剂时一般都是有用的。可溶性(S)CD_4是第一个进行临床测试的进入抑制剂，失败的主要原因是它对艾滋病毒-1的初级分离株表现出较差的中和活性。此外，sCD4在低浓度时表现出促进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)与Ig-α-tp融合，产生了一种重组蛋白，我们称之为D1D2-Ig-α-tp。该重组蛋白的质量大于650千道尔顿(KDa)，存在12个gp120结合位点。12个紧密排列的gp120结合位点的出现应该会提高sCD4与膜CD4竞争病毒粒子相关gp120的能力。此外，该蛋白的极大尺寸应该排除激活的病毒粒子尖峰与靶细胞膜上的CCR5之间的任何相互作用。CCR5是大多数原发HIV感染的重要辅助受体。我们已经在体外表达和鉴定了该蛋白，并发现它与HIV-1主要分离株的囊膜具有极高的亲和力。重要的是，它能有效抑制HIV-1阳性患者外周血单核细胞中的病毒复制。在病毒中和试验中，D1D2-Ig-α-tp抑制病毒复制的水平与现有最有效的中和抗体相当。此外，与单体sCD4不同，在低于最佳浓度的情况下，这种重组蛋白没有表现出促进进入的活性。这些活动中的每一项都表明我们在实现我们的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一种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.