Evolution of anti-filovirus B cell responses and mechanisms of protection

2014年由埃博拉病毒（EBOV）引起的西非疫情凸显了丝状病毒的全球威胁。 近年来，在疫苗和单克隆抗体（MAb）治疗方面取得了重大进展， 包括ZMapp的开发，三种mAb的混合物和两种疫苗平台， 临床试验ZMapp的成功凸显了抗体在预防丝状病毒方面的关键作用 出血热然而，交叉反应性单克隆抗体和疫苗，也将保护对其他成员 丝状病毒科如马尔堡病毒、苏丹病毒和本迪布焦病毒的研究落后。人们对这把钥匙知之甚少 广泛保护性应答的特征，包括丝状病毒表面内的跨种属中和表位 糖蛋白（GP），针对不同表位的抗体协同作用的机制，有效地 中和，决定交叉保护性与物种特异性B细胞应答的演变的特征， 以及在疫苗接种后或自然免疫后引起中和和交叉反应性抗体的程度 exposure.本提案旨在解决这些重要问题。 使用可溶性HIV包膜糖蛋白作为模型抗原，结合B细胞克隆和深度表达， 通过测序，我们已经广泛表征了广泛中和HIV抗体的特征，包括 恒河猴中VH/VL使用和体细胞超突变（SHM）的程度。在这里，我们打算使用一个 类似的方法来理解猕猴中B细胞对丝状病毒抗原的应答的演变。食蟹 和恒河猴是丝状病毒疫苗开发的高度相关模型， 人和猴免疫球蛋白VH基因座之间的差异（91.7±2.2%），也用于B细胞进化的研究 或鉴定治疗性丝状病毒MAb。我们最近进行了两项免疫研究， 在食蟹猴和恒河猴中的丝状病毒抗原，并产生大量外周血 单核细胞（PBMC），骨髓和脾细胞，将用于本提案的深入研究 广泛保护性B细胞应答的表征和新型治疗性单克隆抗体的鉴定。使用 从第一项食蟹猴研究的PBMC中，我们已经鉴定出几种泛埃博拉病毒和泛丝状病毒 抗体，包括在小鼠和豚鼠中交叉中和和保护免受SUDV和EBOV的MAb。 这是针对这两种最不同的埃博拉病毒物种的抗体保护的第一份报告。我们有 还发现了一种新的机制，合作中和某些泛埃博拉病毒单克隆抗体， 这一现象为设计有效的MAb鸡尾酒以及广泛的保护性药物奠定了合理的基础。 疫苗。这项建议有三个具体目标。在目标1中，我们将定义 食蟹猴幼稚IG库，以补充恒河猴上已经存在的知识。新型交叉反应 将使用抗原特异性记忆B细胞分离来自食蟹猴和恒河猴的丝状病毒MAb 分类和克隆。通过NGS抗体组学分析，我们将确定广泛保护性B细胞的特征 包括VH/VL基因使用和SHM的应答。在目的2中，描述了新的表位和作用机制。 将广泛分析交叉反应性MAb，并测试MAb的体内功效。新概念将 开发和评估用于泛丝状病毒疫苗的免疫原改进。在目标3中，抗体 将在接种疫苗的猕猴和来自临床试验的人样品中分析对丝状病毒疫苗的应答。 在西非的试验（NewLink Genetics/Merck）中以及在来自致死性和恢复期的大量血清中， 自然感染的人类。 该多个PI提案将由M博士领导。Javad Aman和Yuxing Li博士在以下方面具有互补的专业知识： 丝状病毒疫苗和免疫治疗剂以及猕猴的免疫遗传学。该团队还包括博士。 John Dye，丝状病毒动物模型的专家，Kartik Karlran博士，丝状病毒受体生物学专家， 博士加里·科宾格（Gary Kobinger），他领导了ZMapp的开发，并密切参与了2014年的疫情应对 在塞拉利昂，托马斯莫纳斯博士，NewLink遗传学，谁将提供从临床材料的访问 在西非进行EBOV VSV疫苗试验。

The 2014 outbreak in West Africa, cause by Ebola virus (EBOV), has highlighted the global threat of filoviruses. Major progress has been made recently towards vaccines and monoclonal antibody (MAb) therapeutics specific for EBOV including development of ZMapp, a cocktail of three mAbs, and two vaccine platforms that advanced to clinical trials. The success of ZMapp has highlighted the critical role of antibodies in protecting against filovirus hemorrhagic fever. However, cross reactive MAbs and vaccines that would also protect against other members of Filoviridae such as Marburg, Sudan, and Bundibugyo viruses are lagging behind. Little is known about the key features of broadly protective responses including cross-species neutralizing epitopes within filovirus surface glycoprotein (GP), the mechanisms by which antibodies against different epitopes synergize for effective neutralization, features that determine the evolution of cross protective versus species specific B cell responses, and the extent to which neutralizing and cross-reactive antibodies are elicited after vaccination or natural exposure. This proposal aims to address these important questions. Using a soluble HIV envelope glycoprotein as model antigen and a combination of B cell cloning and deep sequencing we have extensively characterized the features of broadly neutralizing HIV antibodies including the VH/VL usage and the degree of somatic hypermutations (SHM) in rhesus macaques. Here we intend to use a similar approach to understand the evolution of B cell responses to filovirus antigens in macaques. Cynomolgus and rhesus macaques are highly relevant models for filovirus vaccine development and, given the high homology between the human and monkey immunoglobulin VH loci (91.7±2.2%), also for the study of the B cell evolution or identification of therapeutic filovirus MAbs. We have recently performed two immunization studies using filovirus antigens in cynomolgus and rhesus macaques and generated a large collection of peripheral blood mononuclear cells (PBMC), bone marrow, and splenocytes that will be used in this proposal for in depth characterization of broadly protective B cell responses and identification of novel therapeutic MAbs. Using the PBMC from the first cynomolgus study we have already identified several pan-ebolavirus and pan-filovirus antibodies including MAbs that cross-neutralize and protect against SUDV and EBOV in mice and guinea pigs. This is the first report of antibodies protective against these two most divergent ebolavirus species. We have also discovered a novel mechanism of cooperative neutralization by certain pan-ebolavirus MAbs, a phenomenon that builds a rational basis for design of effective MAb cocktails as well as broadly protective vaccines. This proposal is designed in three specific Aims. In Aim 1, we will define the genetic elements of cynomolgus naïve Ig repertoire to complement the knowledge that already exists on rhesus. Novel cross-reactive filovirus MAbs from cynomolgus and rhesus macaques will be isolated using antigen-specific memory B cell sorting and cloning. Using antibodyome analysis by NGS, we will define the features of broadly protective B cell responses including VH/VL gene usage and SHM. In Aim 2, the epitopes and mechanism of action of the novel cross reactive MAbs will be extensively analyzed and MAbs will be tested for in vivo efficacy. Novel concept will be developed and evaluated for immunogen improvement for pan-filovirus vaccines. In Aim 3, antibody responses to filovirus vaccines will be profiled in vaccinated macaques and in human samples from a clinical trial in West Africa (NewLink Genetics/Merck) as well as in a large number of sera from fatal and convalescent naturally infected humans. This multiple PI proposal will be led by Dr. M. Javad Aman and Dr. Yuxing Li with complementary expertise in filovirus vaccines and immunotherapeutics and immunogenetics of macaques. The team further includes Dr. John Dye with expertise in filovirus animal models, Dr. Kartik Chandran, an expert in filovirus receptor biology, Dr. Gary Kobinger, who led the development of ZMapp and was intimately involved in 2014 outbreak response in Sierra Leone, and Dr. Thomas Monath, NewLink Genetics, who will provide access to material from clinical trials of EBOV VSV vaccine in West Africa.