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

项目摘要/摘要 针对流感病毒表面糖蛋白血凝素(HA)的广谱中和抗体可以提供 对几乎所有流感病毒的保护。然而，广谱中和抗体很少由 疫苗接种，取而代之的是，大多数抗体针对流感病毒HA头部区域的可变表位， 只对少数几种流感病毒株提供狭隘的保护。决定B的基本机制 细胞免疫优势在很大程度上仍不清楚，B细胞的特异性在病毒暴露时会被召回。我们 首次暴露于一种新型流感病毒后，强势诱导了针对四种病毒的抗体 HA的中和表位。然而，反复接触相同的病毒优先推动了招募 针对HA头部不同表位的抗体。值得注意的是，我的研究发现，抗体靶向 广谱中和表位富含多反应性，即单个抗体与多个抗体结合的能力。 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到记忆B细胞池中以提供防御 对抗新的大流行威胁流感病毒。根据我的初步数据，我假设HA表位 特异性影响B细胞的发育、分化和克隆间竞争，从而导致差异 B细胞免疫优势。B细胞的免疫优势可能由三个独立的过程决定：1)B 以广泛中和表位为靶点的细胞可能会经历克隆性删除或由于 多反应性(目标1)，2)靶向广谱中和表位的B细胞分化为短暂的B细胞亚群 与长寿B细胞亚群相反(目标2)，以及3)靶向可变表位的B细胞胜过竞争B细胞 瞄准广泛的中和表位(目标3)。为了测试这些目标，我将使用CRISPR/Cas9来生成B细胞 受体敲入小鼠表达种系版本的针对四个广泛的人的单抗 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细胞的发育 并通过产生混合骨髓嵌合体对每个B细胞受体敲入系的B细胞信号转导潜能进行研究。 在目标2中，我将确定表位特异性是否通过免疫小鼠来塑造B细胞分化潜力 从每个B细胞受体敲入系接受B细胞过继转移并跟踪B细胞分化和 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 通过跟踪生发中心的反应来靶向生发中心内的广泛中和表位 接受针对每个表位的过继转移的B细胞的免疫小鼠。获得的知识 这项研究将为如何广泛诱导中和B细胞提供关键的洞察力，这将有助于 开发一种能对所有流感提供广泛保护的通用流感病毒疫苗 病毒。这个导师奖提供的额外培训将使我能够扩大我的科学和 专业技能，使我的研究独立，并成功地过渡到教员职位。

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.