Rescue of broadly neutralizing mAbs using native trimer

描述(由申请人提供)：广谱中和抗体(BNAbs)可能是有效的HIV-1疫苗所产生的保护性免疫的关键组成部分。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，更好地了解这种广泛的血清中和背后的特殊性将使我们能够识别最理想的靶点，并开发出全面的 疫苗设计策略。新的表位可能特别易于设计或因其异常广泛或有效而具有吸引力。最近，研究人员通过高通量功能筛选和/或从感染的献血者中有针对性地选择表现出广泛血清NAB反应的记忆B细胞来获得各种单克隆性bNAbs(MBNAbs)。然而，这些努力仍然具有挑战性，部分原因是理想的记忆B细胞很少，而且缺乏选择新特异性的真正诱饵。基于天然环境三聚体是NAB的唯一靶标这一前提，我们建议使用仅含有天然环境三聚体的病毒样颗粒(VLP)作为诱饵来获取新的MBNAbs，从而揭示新的脆弱部位。 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。我们将通过三聚体VLP ELISA法确定不同的mBNAb对的结合关系。我们确定的任何拮抗或协同组合都可能影响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。我们将评估一组广泛中和的HIV+血清通过AIM 1中使用的MBNAbs小组抑制三聚体VLP结合的能力。这将使我们能够优先选择其血清表现出新的NAB特异性的供者PBMC中的B细胞选择(AIM 3)。 具体目的3：以荧光三聚体VLP为诱饵拯救MBNAbs。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来标记和检索负责广泛血清中和的MBNAb克隆的方法。我们将优先选择其血清似乎针对目标2中的图谱研究的独特表位的捐赠者。 具体目标4：确定新的MBNAbs的特征。我们将确定新的MBNAbs的特异性、广度和效力。我们将像目标1中一样，研究它们与天然三聚体上其他MBNAb特异性的结合关系。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、片段使用和与最近胚系的差异。 公共卫生相关性：这项建议的公共卫生相关性是为了更好地理解为什么艾滋病毒-1感染者产生的bNAb比迄今接种疫苗所诱导的任何nabb更有效。我们将专注于扩大全新细节的保留范围。这将提供关于HIV-1脆弱部位的新知识，并推动在疫苗设置中引发类似的反应。新抗体在预防或治疗环境中也可能有用，特别是如果它们认识到病毒上的新部位是有效的、广泛的和/或与其他部位协同作用的话。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: The public health relevance of this proposal is to better understand why bNAbs generated in HIV-1-infected individuals are far more effective than any NAbs thus far induced by vaccination. We will focus on expanding the repertoire of completely new specificities. This will provide new knowledge on the vulnerable sites on HIV-1 and impetus to induce similar responses in vaccine setting. New antibodies may also be useful in prophylactic or therapeutic settings, especially if they recognize novel sites on the virus are potent, broad and/or act in synergy with others.