The evolution of dengue virus-reactive circulating antibody repertoire

摘要 四种登革热病毒血清型（DENV 1 - 4）引起人类最重要的蚊媒病毒性疾病， 每年约有5000万登革热病例，全世界有超过30亿人面临感染风险。然而，没有治疗 目前已批准使用，唯一注册的疫苗并不是对所有人群都安全有效。的 这项研究的总体方法是利用我们长期以来的独特纵向样本集， 在尼加拉瓜进行的一项队列研究，旨在解决有关DENV抗体和B细胞免疫学的复杂问题， 相关的流行病学背景。虽然在原发性DENV感染中产生的抗体赋予针对DENV的保护作用， 相同血清型，它们可以保护免受或增强不同DENV的后续感染 血清型;然而，在继发性DENV感染后，个体获得更广泛的保护， DENV血清型，并降低严重疾病的风险。在理解这一点上存在一个关键的差距， 产生和维持广泛中和的循环抗体和B细胞免疫 继发性登革病毒感染我们的初步结果表明，B细胞回忆的扩展有助于 急性继发性登革病毒免疫和后二级记忆B细胞（MBC）显示更广泛的交叉- 血清型中和相比，后初级MBC。我们假设B细胞的募集 从原发性DENV感染到急性继发性循环IgG的记忆，然后大量添加 从继发性DENV感染到持久的血清学和B细胞库的新谱系， 形成持久的继发性登革热后免疫的独特的广泛保护性特征。在这项研究中，我们将 研究在原发性DENV感染期间预先存在的MBC是如何产生的，以及 继发性DENV感染有助于广泛中和。特别是，我们将确定是否广泛 中和抗体来源于预先存在的B细胞区室或在继发性免疫反应中新产生。 感染（目的1），以及它们如何在血清或B细胞库中维持以用于将来的保护（目的2）。 我们将使用创新的病毒抗原分离DENV特异性抗体和B细胞， 最先进的IgG抗体蛋白质组学（Ig-Seq）和B细胞受体测序（BCR-Seq）平台， 生物信息学管道将表达选定的代表性单克隆抗体，并在功能上 使用已建立的标准化中和试验进行评价。我们组建了一个团队 在B细胞生物学、黄病毒病毒学、免疫学和流行病学方面的互补专业知识， 尖端的"抗体组学"血清学和基因组测序技术和分析系统。总体而言， 拟议的研究将开始在细胞和血清学水平上定义抗体的进化- 介导的免疫力，在继发感染时产生针对DENV的广泛中和， 以指导改进的疫苗设计策略。

ABSTRACT The four dengue virus serotypes (DENV1-4) cause the most important mosquito-borne viral disease of humans, with ~50 million dengue cases annually and over 3 billion people worldwide at risk of infection. Yet, no treatment is currently approved for use, and the only registered vaccine is not safe and effective in all populations. The overall approach of this study is to take advantage of unique longitudinal sample sets from our long-standing cohort study in Nicaragua to address complex questions about DENV antibody and B cell immunology in a relevant epidemiological context. While antibodies generated in primary DENV infection confer protection against the same serotype, they can either protect against or enhance a subsequent infection with a different DENV serotype; however, after a secondary DENV infection, individuals acquire broader protection against multiple DENV serotypes, and the risk of severe disease is lowered. There is a critical gap in understanding the generation and maintenance of broadly neutralizing circulating antibodies and B cell immunity stemming from secondary DENV infection. Our preliminary results show that expansion of B cell recall contributes to acute secondary DENV immunity and that post-secondary memory B cells (MBCs) display broader cross- serotype neutralization as compared to post-primary MBCs. We hypothesize that the recruitment of B cell memory from a primary DENV infection into acute secondary circulating IgG, and then substantial addition of new lineages from the secondary DENV infection into long-lasting serological and B cell repertoires, together shape the distinct broadly protective profile of durable post-secondary dengue immunity. In this study, we will investigate how pre-existing MBCs generated during primary DENV infection and newly elicited lineages in secondary DENV infection contribute to broad neutralization. In particular, we will identify whether broadly neutralizing antibodies originate from the pre-existing B cell compartment or are newly elicited in secondary infection (Aim 1), and how they are maintained in serological or B cell repertoires for future protection (Aim 2). We will use innovative viral antigens for isolation of DENV-specific antibodies and B cells coupled with state-of- the-art IgG antibody proteomics (Ig-Seq) and B cell receptor sequencing (BCR-Seq) platforms, with robust bioinformatics pipelines. Selected representative monoclonal antibodies will be expressed and functionally evaluated using established, standardized neutralization assays. We have assembled a team with complementary expertise in B cell biology, flavivirus virology, immunology, and epidemiology, coupled with cutting-edge ‘antibodyomic’ serological and genomic sequencing technologies and analytic systems. Overall, the proposed research will begin to define at the cellular and serological level the evolution of antibody- mediated immunity that generates broad neutralization against DENV upon secondary infection in order to guide improved vaccine design strategies.