Rescue of broadly neutralizing mAbs using native trimer

项目摘要/摘要 广谱中和抗体(BNAbs)可能是保护性免疫的重要组成部分。 一种有效的HIV-1疫苗。自然感染HIV-1的AB反应绝大多数是非中和的。 然而，一小部分患者最终确实会产生异常广泛和有效的bNAb反应。我们 假设更好地了解这一广泛血清背后的特异性 中和将使我们能够识别最理想的目标，并开发出全面的 疫苗设计策略。新的表位可能特别容易设计或通过以下方式吸引人 异常广泛的或有力的。最近，研究人员通过HIGH获得了各种单克隆性的bNAbs(MbNAbs) 吞吐量功能筛查和/或通过靶向选择来自受感染捐赠者的记忆B细胞 表现出广泛的血清NAB反应。然而，这些努力仍然具有挑战性，部分原因是 理想的记忆B细胞，以及缺乏选择新的特异性的真正诱饵。基于这样一个前提： 原生环境三聚体是NABS的专属靶标，在此我们建议使用携带病毒样颗粒(VLP) 只有本地环境三聚体作为诱饵来检索新的MBNAbs，这将揭示新的易受攻击的部位。 我们的具体目标是： 具体目的1：研究天然三聚体VLP上的单克隆性bNAb关系。我们将决定 用三聚体VLP-ELISA法研究不同mBNAb对的结合关系。任何对抗性或协同性 我们确定的组合可能会影响MBNAb组合的治疗或预防应用。 选定的MBMAb组合将在中和协同试验中进一步研究。 具体目标2：用天然三聚体VLP定位广谱中和血清的特异性。我们会 评估一组广泛中和HIV+血清抑制三聚体VLP结合的能力 目标1中使用的MBNAbs。这将使我们能够优先从其血清中的供者PBMC中选择B细胞(目标3 展示新奇的NAB特性。 具体目的3：以荧光三聚体VLP为诱饵拯救MBNAbs。与一位专家合作者， 我们将开发利用荧光标记的三聚体VLP作为诱饵和探针供体PBMC进行标记的方法 并回收负责广泛血清中和的mBNAb克隆。我们将优先考虑其血清 似乎以目标2中的图谱研究中的独特表位为目标。 具体目标4：确定新的MBNAbs的特征。我们将确定新的特定性、广度和效力 MBNAbs。我们将检查它们与本地三聚体上的其他mBNAb特异性的结合关系，如 目的1.为了更好地了解它们的个体发育，我们还将研究它们的序列、片段使用和 从最近的生殖系分化而来。

Project Summary/Abstract Broadly neutralizing antibodies (bNAbs) may be crucial component of the protective immunity conferred by an effective HIV-1 vaccine. Ab responses in natural HIV-1 infection are overwhelmingly non-neutralizing. However, a fraction of patients do eventually develop exceptionally broad and potent bNAb responses. We hypothesize that a better understanding of the specificities that underlie this broad serum neutralization will enable us to identify the most desirable targets and develop a full spectrum of vaccine design strategies. New epitopes may be particularly amenable to design or attractive by being unusually broad or potent. Researchers have recently accessed various monoclonal bNAbs (mbNAbs) by high throughput functional screening and/or by targeted selection of memory B cells from infected donors who exhibit broad serum NAb responses. However, these efforts remain challenging, in part due to the rarity of desirable memory B cells and the lack of authentic baits to select new specificities. Based on the premise that native Env trimers are the exclusive targets of NAbs, here we propose using virus-like particles (VLPs) bearing only native Env trimers as baits to retrieve novel mbNAbs that will illuminate novel sites of vulnerability. Our Specific Aims are: Specific Aim 1: To investigate monoclonal bNAb relationships on native trimer VLPs. We will determine the binding relationships of various mbNAb pairs by trimer VLP ELISA. Any antagonistic or synergistic combinations we identify could impact the therapeutic or prophylactic applications of mbNAb combinations. Selected mbMAb combinations will be further investigated in neutralization synergy assays. Specific Aim 2: To map the specificities of broadly neutralizing sera using native trimer VLPs. We will evaluate the ability of a panel of broadly neutralizing HIV+ sera to inhibit trimer VLP binding by the panel of mbNAbs used in Aim 1. This will allow us to prioritize B cell selections (Aim 3) from donor PBMCs whose sera exhibit novel NAb specificities. Specific Aim 3: To rescue mbNAbs using fluorescent trimer VLPs as baits. With an expert collaborator, we will develop methods to use fluorescently labeled trimer VLPs as baits and probe donor PBMCs to label and retrieve mbNAb clones responsible for broad serum neutralization. We will prioritize donors whose sera appear to target unique epitopes from mapping studies in Aim 2. Specific Aim 4: To characterize new mbNAbs. We will determine the specificity, breadth and potency of new mbNAbs. We will examine their binding relationships with other mbNAb specificities on the native trimer, as in Aim 1. To better understand their ontogeny, we will also examine their sequences, segment usage and divergence from nearest germline.