Kinetic Determinants of HIV Neutralization

HIV 中和的动力学决定因素

• 批准号: 7929311
• 负责人: Gregory B Melikian
• 金额: $ 23.25万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-15 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7929311
• 关键词: Affect; Antibodies; Attenuated; Binding; Binding Sites; Biological Assay; CD4 Positive T Lymphocytes; Cell fusion; Cell membrane; Cell surface; Cells; Complex; Defensins; Dynamin; Endocytosis; Endosomes; Epithelial; Epitopes; Future; Glycoproteins; Goals; HIV; HIV-1; Human; Image; Immunologic Surveillance; Infection; Intervention; Kinetics; Knowledge; Lymphoid Cell; Masks; Measurement; Measures; Mediating; Membrane; Membrane Fusion; Molecular Conformation; Monitor; Outcome; Pathway interactions; Physiological; Predisposition; Process; Reaction; Relative (related person); Role; Route; Site; Structure; Surface; T-Lymphocyte; Testing; Therapeutic; Time; Viral; Virus; Work; density; design; in vivo; inhibitor/antagonist; neutralizing antibody; novel; novel strategies; prevent; public health relevance; receptor; research study; residence; response; tool; uptake

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.

描述（由申请人提供）：人类免疫缺陷病毒（HIV）通过通过病毒包膜（ENK）糖蛋白介导的过程将其包膜膜与细胞膜融合与细胞膜来感染细胞。在融合反应期间，ENV经历了在与CD4和共感受器的顺序相互作用上触发的广泛构象变化。这些构象变化与抗体通常无法访问的保守功能重要域的短暂暴露有关。尽管有这种暴露，但对这些结构域的中和抗体很少见。对抗体中和艾滋病毒的能力的决定因素知之甚少。除了似乎限制了对关键ENV表位的空间因子外，还有证据表明，与CD4诱导的（CD4I）表位结合的抗体通过短暂暴露这些结构域进行了动力限制。我们最近表明，HIV-1无法与质膜融合，并通过动力蛋白依赖性内吞作用进入细胞。内体融合发生在病毒摄取后长时间延迟后发生，这表明相对较快的HIV-1内部化可以保护病毒免受抗体和靶向ENV中间体的融合抑制剂的影响。我们假设CD4I抗体的功效部分取决于细胞表面上适当的ENV表位的时间。为了检验这一假设，我们将增加ENV中间体的寿命，并确定该干预措施是否使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通过内吞作用是否进入原发性CD4+ T细胞（AIM 1）。对这些细胞的首选HIV-1进入途径的知识将指导我们为增强CD4I抗体功效的努力。具体而言，CD4结合状态的寿命将通过降低共感受器的密度（AIM 2A）来延长，而CD4/CORECEPTOR结合状态的停留时间将通过抑制生产性HIV-1内吞作用（AIM 2B）延长。通过新开发的动力学测定，允许通过CD4和CD4/CD4/CORECEPTOR结合的状态在细胞表面上测量HIV-1进展，这些关键中间体的寿命将被确定并与CD4I抗体阻断融合的能力相关。预计这些研究将阐明动力学因子在HIV-1中和，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）通过通过病毒包膜（ENK）糖蛋白介导的过程将其包膜与宿主细胞膜融合来感染细胞。尽管在HIV融合期间，保守环境的瞬时暴露，但识别这些区域的抗体中和抗体很少。为了测试抗体与保守环境结合的假设由于其短暂暴露而受到限制，我们将稳定ENV在细胞表面的中间构象，并确定该干预措施是否增强了针对这些区域的中和抗体活性。