Investigating the development and clonal dynamics of broadly neutralizing B cells against influenza viruses

项目总结/摘要 针对流感病毒表面糖蛋白血凝素（HA）的广泛中和抗体可以提供 几乎所有流感病毒的保护。然而，广泛中和抗体很少由 相反，大多数抗体靶向流感病毒HA头部结构域的可变表位， 仅提供针对少数流感病毒株的有限保护。决定B的基本机制 细胞免疫优势，即B细胞特异性在病毒暴露后被召回，在很大程度上仍然是未知的。我们 首次接触一种新型流感病毒， HA的中和表位。然而，反复暴露于相同的病毒优先驱动招聘 的抗体靶向HA头部的可变表位。值得注意的是，我的研究发现， 广泛中和表位富含多反应性，即单个抗体结合多个表位的能力。 分子上不同的抗原，包括外来抗原和自身抗原。此外，多反应性幼稚B细胞 靶向广泛中和表位优先选择进入记忆B细胞库以提供防御 对抗新的大流行威胁流感病毒。基于我的初步数据，我假设HA表位 特异性影响B细胞发育、分化和克隆间竞争，从而导致差异 在B细胞免疫优势中。B细胞免疫优势可由三个独立过程决定：1）B 靶向广泛中和表位的细胞可能经历克隆缺失或由于被 多反应性（Aim 1），2）靶向广泛中和表位的B细胞分化为短寿命B细胞亚群 与长寿的B细胞亚群相反（Aim 2），和3）靶向可变表位的B细胞胜过B细胞 靶向广泛中和表位（Aim 3）。为了测试这些目标，我将使用CRISPR/Cas9来产生B细胞。 受体敲入小鼠表达人单克隆抗体的种系版本， HA的中和表位和HA的两个可变表位。为了测试目标1，我将评估B细胞发育 和每个B细胞受体敲入系的B细胞信号传导潜力。 在目标2中，我将通过免疫小鼠来确定表位特异性是否影响B细胞分化潜能， 接受来自每个B细胞受体敲入系的B细胞过继转移并跟踪B细胞分化， 亲和力成熟在目标3中，我将确定靶向可变表位的B细胞是否胜过B细胞 通过追踪HA中的germinal中心应答，靶向germinal中心内的广泛中和表位， 已接受靶向每个表位的过继转移的B细胞的免疫小鼠。获得的知识 这项研究的结果将为如何诱导广泛中和的B细胞提供重要的见解，这将有助于 在开发一种通用流感病毒疫苗，可以提供广泛的保护， 病毒这个指导奖提供的额外培训将使我能够扩大我的科学和 专业技能，导致我的研究独立性和成功过渡到教师的位置。

PROJECT SUMMARY/ABSTRACT Broadly neutralizing antibodies against the influenza virus surface glycoprotein hemagglutinin (HA) can provide protection from nearly all influenza viruses. However, broadly neutralizing antibodies are rarely induced by vaccination and instead, most antibodies target variable epitopes of the influenza virus HA head domain that only provide narrow protection against a few influenza virus strains. The fundamental mechanisms dictating B cell immunodominance, which B cell specificities are recalled upon virus exposure, remain largely unknown. We identified that first exposure to a novel influenza virus robustly induced antibodies against four broadly neutralizing epitopes of HA. However, repeated exposure to the same virus preferentially drove the recruitment of antibodies targeting variable epitopes of the HA head. Notably, my studies identified that antibodies targeting broadly neutralizing epitopes are enriched for polyreactivity, the ability of a single antibody to bind to multiple molecularly distinct antigens, including foreign and self-antigens. Furthermore, polyreactive naïve B cells targeting broadly neutralizing epitopes are preferentially selected into the memory B cell pool to provide defense against novel pandemic-threat influenza viruses. Based on my preliminary data, I hypothesize that HA epitope specificity influences B cell development, differentiation, and inter-clonal competition, which leads to differences in B cell immunodominance. B cell immunodominance may be dictated by three independent processes: 1) B cells targeting broadly neutralizing epitopes may undergo clonal deletion or become anergic as a result of being polyreactive (Aim 1), 2) B cells targeting broadly neutralizing epitopes differentiate into short-lived B cell subsets as opposed to long-lived B cell subsets (Aim 2), and 3) B cells targeting variable epitopes outcompete B cells targeting broadly neutralizing epitopes (Aim 3). To test these aims, I will use CRISPR/Cas9 to generate B cell receptor knock-in mice expressing the germline version of human monoclonal antibodies targeting four broadly neutralizing epitopes of HA and two variable epitopes of HA. To test Aim 1, I will evaluate B cell development and B cell signaling potential of each B cell receptor knock-in line by generating mixed bone marrow chimeras. In Aim 2, I will determine if epitope specificity shapes B cell differentiation potential by immunizing mice that receive a B cell adoptive transfer from each B cell receptor knock-in line and tracking B cell differentiation and affinity maturation. In Aim 3, I will determine whether B cells targeting a variable epitope outcompete B cells targeting a broadly neutralizing epitope within the germinal center by tracking germinal center responses in HA- immunized mice that have received adoptively transferred B cells targeting each epitope. Knowledge gained from this research will provide critical insight into how broadly neutralizing B cells can be induced, which will aid in the development of a universal influenza virus vaccine that can provide broad protection against all influenza viruses. The additional training afforded by this mentored award will enable me to expand my scientific and professional skillsets, leading to my research independence and successful transition into a faculty position.