Functions of human C. difficile-specific memory B cell-derived monoclonal antibodies

项目概要/摘要（项目2） 艰难梭菌病复发是一个严重的问题，因为严重程度随着每一轮的治疗而增加。 感染并将局部肠道疾病转化为全身性致命疾病。我们缺乏很好的理解 感染的免疫反应，这可以说是缺乏或错误的方向，如反复发作的 感染血清IgG抗体（Abs）中和C.艰难梭菌（TcdB）是最好的相关性 保护。理想情况下，感染会刺激初级毒素中和抗体反应，并诱导毒素- 特异性记忆B细胞（BCRs），其可以对病原体快速响应并产生新的Ab分泌细胞。 然而，在小鼠模型中和在对人BAE的分析中，感染导致的应答是显性的， IgM+细胞，尽管一些IgG+和伊加+细胞是明显的。人TcdB的分离和单细胞条形码化 测序和库分析显示IgG+和伊加+细胞经历了特异性的BMPs， 体细胞超突变，并具有广泛的可变基因使用，代表了几个独特的B细胞克隆。 从选定的IgG 1基因序列产生的单克隆抗体（mAb）显示对TcdB具有中等亲和力 和差的TcdB中和。这项工作引导我们提出了一个假设，即TcdB特异性 C.艰难梭诱导人B细胞记忆区室具有可变的 中和毒素的能力。在具体目标1中，我们将测量TcdB特异性Bmem衍生的 单克隆抗体对宿主细胞中毒机制的研究。我们将从我们数据库中的基因序列中产生mAb 并招募新的志愿者，以扩大BMEM剧目的数量。我们将确定BERO的影响- 衍生的单克隆抗体对控制宿主细胞中毒的机制，并提供了一个全面的看法， 在C.艰难感染在特定 目的2：我们将测试源自骨髓基质细胞的mAb保护免受活病原体攻击的能力， 确定运输到肠道的机制。我们发现新生儿Fc受体（FcRn）是 将免疫诱导的循环IgG递送至肠道并保护免受C.艰难，而 腹膜内递送至受体小鼠绕过了FcRn要求。我们将通过腹腔注射mAb， B6小鼠以确定人Bmem衍生的mAb是否在体内具有保护性。我们将使用FcRn-/-小鼠表达 人FcRn和辅助分子β2微球蛋白（hβ 2 M）转基因（hFcRn：hβ 2 M），以确定 循环mAb保护的hFcRn依赖性。目标2将提供关于BMEM编码是否 mAb在体内具有保护作用，并决定它们如何到达肠道。项目2具有高度的相关性， 公共卫生我们目前对C.人类受试者中的艰难梭菌缺乏 必要的机械见解，以解释为什么病人容易复发感染。我们的工作将 确定是否BELCADE隔室不充分编码保护性抗体，并揭示功能性抗体， 保护性与非保护性Bronchial细胞的特性，并为疫苗接种策略提供信息。

PROJECT SUMMARY / ABSTRACT (Project 2) Clostridioides difficile disease recurrence is a serious problem because severity increases with each round of infection and converts a regional enteric disease into a systemic fatal disease. We lack a good understanding of the immune response to infection, which arguably is lacking or mis-directed, as evidenced by recurrent infection. Serum IgG antibodies (Abs) that neutralize toxin B secreted by C. difficile (TcdB) is the best correlate of protection. Ideally, infection would stimulate primary toxin-neutralizing Ab responses as well as induce toxin- specific memory B cells (Bmem) that can respond rapidly to the pathogen and generate new Ab-secreting cells. However, in mouse models and in analysis of human Bmem, infection results in a response which is dominated by IgM+ cells, although some IgG+ and IgA+ cells are evident. Isolation and single cell barcoding of human TcdB- specific Bmem followed by sequencing and repertoire analysis revealed that IgG+ and IgA+ cells had undergone somatic hypermutation and had breadth of variable gene usage, representing several unique B cell clones. Production of monoclonal Abs (mAbs) from selected IgG1 gene sequences revealed moderate affinity for TcdB and poor TcdB neutralization in one in vitro assay. This work has guided us to a hypothesis that TcdB-specific IgG and IgA encoded by the C. difficile-induced human B cell memory compartment have variable capacity for toxin-neutralization. In Specific Aim 1 we will measure the impact of TcdB-specific Bmem-derived mAbs on the mechanisms of host cell intoxication. We will produce mAbs from gene sequences in our database and recruit new volunteers to expand the number of Bmem-repertoires. We will determine the impact of Bmem- derived mAbs on the mechanisms controlling host cell intoxication and provide a comprehensive view of the functions of human Bmem cell-encoded TcdB-specific Abs in individuals following C. difficile infection. In Specific Aim 2: We will test the ability of Bmem cell-derived mAbs to protect against a live pathogen challenge and determine mechanism of transport to the gut. We showed that the neonatal Fc receptor (FcRn) was required for delivery of immunization-induced circulating IgG to the gut and protection against C. difficile, whereas intraperitoneal delivery to recipient mice bypassed the FcRn requirement . We will deliver intraperitoneal mAb to B6 mice to determine if human Bmem-derived mAbs are protective in vivo. We will use FcRn-/- mice expressing the human FcRn and accessory molecule β2 microglobulin (hβ2M) transgenes (hFcRn:hβ2M ) to determine hFcRn dependence for protection by circulating mAb. Aim 2 will provide critical data on whether Bmem-encoded mAbs are protective in vivo and determine how they reach the gut. Project 2 has a high degree of relevance to public health. Our current understanding of the humoral immune response to C. difficile in human subjects lacks the necessary mechanistic insights to explain why patients are prone to recurrent infection. Our work will determine if the Bmem compartment insufficiently encodes protective Abs and also reveal the functional properties of protective versus non-protective Bmem cells and inform vaccination strategies.