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

研究针对流感病毒的广泛中和 B 细胞的发育和克隆动态

• 批准号: 10660297
• 负责人: Jenna Guthmiller
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-08 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10660297
• 关键词: Adoptive Transfer; Adult; Affinity; Antibodies; Antibody Response; Antigens; Autoantigens; Award; B cell differentiation; B-Cell Antigen Receptor; B-Cell Development; B-Lymphocyte Subsets; B-Lymphocytes; Binding; Biology; Bone Marrow; CRISPR/Cas technology; Cell physiology; Cessation of life; Chimera organism; Clonal Deletion; Clone Cells; Common Epitope; Data; Development; Disadvantaged; Dissection; Epitopes; Exposure to; Faculty; Goals; Head; Hemagglutinin; Human; Immunity; Immunization; Immunize; Influenza A Virus, H1N1 Subtype; Influenza Hemagglutinin; Influenza vaccination; Knock-in; Knock-in Mouse; Knowledge; Membrane Glycoproteins; Memory B-Lymphocyte; Mentors; Molecular; Monoclonal Antibodies; Mus; Mutation Analysis; Positioning Attribute; Process; Production; Public Health; Receptor Signaling; Regimen; Research; Shapes; Signal Transduction; Specificity; Structure of germinal center of lymph node; Testing; Training; Vaccination; Viral; Virus; Zoonoses; adoptive B cell transfer; anergy; base; experimental study; exposed human population; human monoclonal antibodies; in vivo; influenza infection; influenza virus strain; influenzavirus; insight; memory recall; neutralizing antibody; novel; novel vaccines; pandemic disease; recruit; response; universal influenza vaccine; vaccine platform

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.

项目摘要/摘要 针对影响Za病毒表面糖蛋白血凝素（HA）的广泛中和抗体可以提供 保护几乎所有影响力病毒。但是，很少诱导广泛中和抗体 相反，大多数抗体靶向影响病毒HA头域的可变表位， 仅提供对一些影响病毒菌株的狭窄保护。决定B的基本机制 病毒暴露后召回了B细胞规格的细胞免疫优势，在很大程度上尚不清楚。我们 确定首次暴露于新型病毒会强烈诱导针对四种的抗体 中和HA的表位。但是，重复接触同一病毒优先驱动招募 靶向HA头的变量表位的抗体的。值得注意的是，我的研究表明靶向抗体 广泛中和的表位富含多反应性，单一抗体与多种结合的能力 分子不同的抗原，包括外抗原和自我抗原。此外，多反应性幼稚的B细胞 靶向广泛中和的表位优选到记忆B细胞池中以提供防御 反对新的大流行威胁影响病毒。根据我的初步数据，我假设HA表位 特异性影响B细胞的发展，分化和连锁间竞争，从而导致差异 在B细胞免疫主持中。 B细胞免疫率可能由三个独立过程决定：1）b 靶向广泛中和表位的细胞可能会经历克隆缺失或因其而变为厌食症。 多反应性（AIM 1），2）靶向广泛中和表位的B细胞分化为短寿命的B细胞子集 与长寿命B细胞子集（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生成B单元 表达人类单克隆抗体的种系版本的受体敲入小鼠靶向四种抗体 HA的中和表位和HA的两个可变表位。要测试目标1，我将评估B细胞的开发 通过产生混合骨髓嵌合体，每个B细胞受体敲入线的B细胞信号传导电位。 在AIM 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细胞接收器的敲入线并跟踪B细胞分化和 亲和力成熟。在AIM 3中，我将确定靶向可变表位的B细胞是否超过B细胞 通过跟踪HA-中的生发中心反应来瞄准生发中心内广泛中和的表位 接收到针对每个表位的适当转移的B细胞的免疫小鼠。知识获得了 从这项研究中，将提供有关如何诱导B细胞的广泛中和B细胞的关键见解，这将有助于 在开发一种普遍影响力的病毒疫苗中，可以为所有影响力提供广泛的保护 病毒。该审判奖提供的额外培训将使我能够扩展我的科学和 专业技能，导致我的研究独立性并成功过渡到教师职位。