Rescue of broadly neutralizing mAbs using native trimer

项目概要/摘要 广泛中和抗体（bNAbs）可能是保护性免疫的关键组成部分， 有效的HIV-1疫苗。自然HIV-1感染中的抗体应答绝大多数是非中和性的。 然而，一小部分患者最终确实发展出异常广泛和有效的bNAb应答。我们 假设更好地理解这种广泛血清的特异性 中和将使我们能够确定最理想的目标，并制定一个全方位的 疫苗设计策略。新的表位可能特别适合设计或具有吸引力， 异常广泛或有效。研究人员最近通过高通量测序获得了各种单克隆bNAb（mbNAb）。 通量功能筛选和/或通过靶向选择来自受感染供体的记忆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细胞和缺乏真正的诱饵来选择新的特异性。为前提 天然Env三聚体是NAb的唯一靶点，在此我们建议使用携带病毒样颗粒（VLP） 仅天然Env三聚体作为诱饵以检索将阐明新的脆弱性位点的新的mbNAb。 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bNAb关系。我们将确定 通过三聚体VLP ELISA测定各种mbNAb对的结合关系。任何拮抗或协同作用 我们鉴定的这些组合可能影响mbNAb组合的治疗性或预防性应用。 将在中和协同试验中进一步研究选定的mbMAb组合。 具体目的2：使用天然三聚体VLP绘制广泛中和血清的特异性。我们将 评价一组广泛中和的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考虑从供体PBMC中选择B细胞（目标3）， 显示新的NAb特异性。 具体目标3：使用荧光三聚体VLP作为诱饵拯救mbNAb。与专家合作， 我们将开发使用荧光标记的三聚体VLP作为诱饵和探针供体PBMC来标记的方法， 并回收负责广泛血清中和的mbNAb克隆。我们会优先考虑那些 似乎靶向来自Aim 2中的定位研究的独特表位。 具体目标4：表征新的mbNAb。我们将确定新的具体性，广度和效力 mbNAbs。我们将检查它们与天然三聚体上的其他mbNAb特异性的结合关系，如在 目标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 与最近的生殖系的分歧。

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.