Identifying human antibodies that potently neutralize cell-to-cell HIV infection

描述（申请人提供）：开发产生中和抗体的人类免疫缺陷病毒1型（HIV-1）疫苗的努力集中在阻断无细胞病毒传播的抗体上，然而，最近的体外研究越来越清楚地表明，T细胞通过一种称为病毒学突触的结构传播HIV是一种更有效的感染模式。 我们最近的研究表明，能够中和无细胞HIV-1的抗体在中和细胞相关HIV-1方面的效果往往明显较差。 在检测的抗体中，我们发现 与同源无细胞病毒攻击相比，HIV感染患者血清在阻断细胞相关HIV-1方面弱得多。 在通过病毒突触感染期间，病毒进入受体Env首先作为细胞粘附受体发挥作用，其次在转移到靶CD 4 T细胞中后，它作为病毒膜融合装置发挥作用。 Env在此过程中的构象调节可能有助于细胞相关的HIV抵抗许多中和反应。 本研究的一般假设是，细胞表面上的病毒进入受体Env与激发最强免疫应答的Env在构象上不同。 如果病毒学突触介导的感染是体内病毒传播的主要模式，那么靶向细胞表面或病毒体表面上Env的更多“天然”构象的疫苗可能证明在阻断传播方面更有效。 在这个项目中，我们将采用一种新的方法来测量抗原特异性B细胞， Env的天然构象 在这种方法中，荧光HIV感染的细胞或携带GFP的重组HIV-1颗粒将作为抗原探针来检测患者体内的天然HIV结合B细胞。 我们将把自然HIV结合记忆B细胞的频率与每个患者的细胞间或无细胞感染的中和抗体滴度相关联。 表达来自不同进化枝的Env的荧光vral克隆将测量患者中病毒结合应答的宽度。我们将进一步测试未成熟的病毒颗粒，这些病毒颗粒可能以与细胞表面更相似的构象显示Env，以确定这些抗体是否可以更好地中和细胞间感染。来自HIV特异性记忆B细胞的单个免疫球蛋白（IG）基因将与来自西奈山治疗抗体开发中心（CCED）的共同研究者使用单细胞克隆方法从患者细胞中克隆。 我们将功能性地筛选所产生的单克隆抗体，以寻找那些有效抑制细胞间介导的感染的单克隆抗体。 具体目标如下。 目标1：使用荧光感染细胞和HIV Gag-iGFP颗粒定量慢性HIV-1感染患者中的HIV结合记忆B细胞，并将抗原特异性B细胞的这种直接测量与血清抗体中和无细胞或细胞间感染的能力相关联。 目标二：从单个HIV结合B细胞直接克隆单克隆抗体，并检查中和细胞间和无细胞病毒感染的广度和效力。

DESCRIPTION (provided by applicant): Efforts to develop an human immunodeficiency virus type 1 (HIV-1) vaccine that generate neutralizing antibodies have focused on antibodies that block cell-free viral transmission, yet it is increasingly clear from rcent in vitro studies that direct T cell-to-T cell transmission of HIV through a structures called virological synapses is much more efficient mode of infection. Our recent studies suggest that antibodies that can neutralize cell-free HIV-1 are often significantly less effective at neutralizing cell-associated HIV-1. Among antibodies tested, we have found that HIV-infected patient sera can be much weaker at blocking cell-associated HIV-1 when compared to homologous cell-free virus challenge. During infection through virological synapses, the viral entry receptor, Env, first functions as a cell-adhesion receptor, and secondly after a transfer into target CD4 T cells, it functions as viral membrane fusion apparatus. The conformational regulation of Env during this process may help cell-associated HIV to resist many neutralizing responses. A general hypothesis of this study is that the viral entry receptor, Env, on the surface of cells is conformationally distinct from the Env that elicits the strongest immune responses. If virological synapse-mediated infection is a predominant mode of viral dissemination in vivo, then vaccines that target more "native" conformations of Env on the cell-surface or virion surface, may prove more effective at blocking transmission. In this project we will employ a new method to measure antigen specific B cells that targets native conformations of Env. In this approach, fluorescent HIV-infected cells or recombinant GFP-carrying HIV-1 particles will function as antigenic probes to detect native HIV-binding B cells in patients. We will correlate the frequency of native HIV-binding memory B cells with each patient's neutralizing antibody titers against cell-to-cell or cell-free infections. Fluorescent vral clones expressing Env from different clades will measure the breadth of virus-binding responses in patients. We will further test immature virus particles that may display Env in conformations more similar to those on the surface of cells to see if these identify unique antibodies that can better neutralize cell-to-cell infection. Individual immunoglobulin (Ig) genes from HIV-specific memory B cells will be cloned from patient cells using single cell cloning approaches with coinvestigators from the Mount Sinai Center for Therapeutic Antibody Development (CTAD). We will functionally screen the resulting monoclonal antibodies for those that potently inhibit cell-to-cell mediated infection. The specific aims are as follows. Aim 1: o quantify HIV-binding memory B cells in chronically HIV-1 infected patients using fluorescent infected cells and HIV Gag-iGFP particles and to correlate this direct measure of antigen specific B cells with the ability of serum antibodies to neutralize cell-free or cell-to-cell infecion. Aim 2: To directly clone monoclonal antibodies from individual HIV-binding B cells and to examine the breadth and potency in neutralizing cell-to-cell and cell-free viral infection.