Kinetic Determinants of HIV Neutralization

描述(申请人提供)：人类免疫缺陷病毒(HIV)通过病毒包膜(Env)糖蛋白介导的过程将其包膜与细胞膜融合，从而感染细胞。在融合反应中，Env经历了广泛的构象变化，这些变化是由与CD4和辅助受体的顺序相互作用触发的。这些构象变化与抗体通常无法接触到的保守的功能重要结构域的瞬时暴露有关。尽管有这种暴露，但针对这些区域的中和抗体很少见。抗体中和艾滋病毒能力的决定因素还知之甚少。除了限制获得关键环境抗原表位的空间因素外，还有证据表明，抗体与CD4诱导的(CD4i)表位的结合受到这些区域的短暂暴露的动力学限制。我们最近发现，HIV-1不能与质膜融合，并通过动力蛋白依赖的内吞作用进入细胞。内体融合发生在病毒摄取后很长一段时间后，这表明相对较快的HIV-1内化可以保护病毒免受针对Env中间体的抗体和融合抑制剂的攻击。我们假设CD4i抗体的有效性部分取决于细胞表面适当的环境抗原表位暴露的时间。为了验证这一假设，我们将延长Env中间体的寿命，并确定这种干预是否会使HIV-1对中和抗体敏感。为了确定这种方法在体内增强CD4i抗体活性的相关性，我们将首先验证HIV-1通过内吞作用进入原代CD4+T细胞(目标1)。对HIV-1进入这些细胞的首选途径的了解将指导我们努力提高CD4i抗体的疗效。具体地说，通过降低辅助受体的密度将延长CD4结合状态的寿命(目标2a)，而通过抑制产生HIV-1的内吞作用将延长处于CD4/辅助受体结合状态的停留时间(目标2b)。通过新开发的动力学分析方法，可以通过细胞表面的CD4和CD4/辅助受体结合状态对HIV-1进展进行时间分辨测量，这些关键中间体的寿命将被确定，并与CD4i抗体阻止融合的能力相关联。这些研究有望阐明动力学因素在HIV-1中和过程中的作用，并验证旨在增强CD4i抗体活性的新方法。 公共卫生相关性：人类免疫缺陷病毒(HIV)通过病毒包膜(Env)糖蛋白介导的过程将其包膜与宿主细胞膜融合，从而感染细胞。尽管在HIV融合过程中，保守的Env区域会短暂暴露，但识别这些区域的中和抗体很少见。为了验证抗体与保守的Env区域的结合由于短暂暴露而受到限制的假设，我们将稳定细胞表面Env的中间构象，并确定这种干预是否增强了针对这些区域的抗体的中和活性。

DESCRIPTION (provided by applicant): The Human Immunodeficiency Virus (HIV) infects cells by fusing its envelope membrane with the cell membrane through a process that is mediated by the viral envelope (Env) glycoprotein. During the fusion reaction, Env undergoes extensive conformational changes that are triggered upon sequential interactions with CD4 and coreceptors. These conformational changes are associated with transient exposure of conserved functionally important domains that are normally inaccessible to antibodies. In spite of this exposure, neutralizing antibodies against these domains are rare. The determinants of the ability of antibodies to neutralize HIV are poorly understood. In addition to steric factors that appear to limit the access to critical Env epitopes there is evidence that antibody binding to CD4-induced (CD4i) epitopes is kinetically restricted by brief exposure of these domains. We have recently shown that HIV-1 is unable to fuse with the plasma membrane and enters cells via dynamin-dependent endocytosis. Endosomal fusion occurs after a long delay following the virus uptake, suggesting that the relatively quick HIV-1 internalization could protect the virus from antibodies and fusion inhibitors targeting Env intermediates. We hypothesize that the efficacy of CD4i antibodies is determined in part by the time of exposure of proper Env epitopes on the cell surface. To test this hypothesis, we will increase the lifetime of Env intermediates and determine whether this intervention sensitizes the HIV-1 to neutralizing antibodies. To establish the relevance of this approach to enhancing the activity of CD4i antibodies in vivo, we will first verify that HIV-1 enters primary CD4+ T cells via endocytosis (Aim 1). The knowledge of the preferred HIV-1 entry route into these cells will guide our efforts to enhance the efficacy of CD4i antibodies. Specifically, the lifetime of the CD4-bound state will be extended by lowering the density of coreceptors (Aim 2a), whereas the residence time in the CD4/coreceptor-bound state will be prolonged by inhibiting productive HIV-1 endocytosis (Aim 2b). Through the newly developed kinetic assays that permit the time-resolved measurement of the HIV-1 progression through CD4- and CD4/coreceptor-bound states on the cell surface, the lifetimes of these key intermediates will be determined and correlated with the ability of CD4i antibodies to block fusion. These studies are expected to elucidate the role of kinetic factors in HIV-1 neutralization and to validate novel approaches aimed at enhancing the activity of CD4i antibodies. PUBLIC HEALTH RELEVANCE: The Human Immunodeficiency Virus (HIV) infects cells by fusing its envelope with the host cell membrane through a process that is mediated by the viral envelope (Env) glycoprotein. In spite of the transient exposure of conserved Env regions during HIV fusion, neutralizing antibodies recognizing these regions are rare. To test the hypothesis that antibody binding to conserved Env regions is restricted due to their brief exposure, we will stabilize intermediate conformations of Env on the cell surface and determine whether this intervention enhances the neutralizing activity of antibodies against these regions.