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

人艰难梭菌特异性记忆 B 细胞来源的单克隆抗体的功能

• 批准号: 10625176
• 负责人: Mark L Lang
• 金额: $ 44.85万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-25 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10625176
• 关键词: Affinity; Antibodies; Antibody Response; Antigens; B cell repertoire; B-Lymphocytes; Bar Codes; Binding; Biological Assay; Breeding; Bypass; Cell Compartmentation; Cells; Clone Cells; Clostridium difficile; Data; Databases; Dependence; Disease; Enteral; Fc Receptor; Generations; Genes; Genomics; Goals; Guanosine Triphosphate Phosphohydrolases; Human; IgG1; IgG2; Immune response; Immunization; Immunoglobulin A; Immunoglobulin G; Immunoglobulin M; Immunoglobulin Somatic Hypermutation; Immunoglobulin-Secreting Cells; Immunologic Memory; In Vitro; Individual; Infection; Intoxication; Intravenous; Life; Measures; Mediating; Memory B-Lymphocyte; Modeling; Monoclonal Antibodies; Mus; Oklahoma; Outcome; Pathology; Patients; Pattern; Plasma Cells; Production; Property; Public Health; Recurrence; Recurrent disease; Relapse; Serum; Severities; Severity of illness; Symptoms; Technology; Testing; Therapeutic Monoclonal Antibodies; Toxin; Transgenes; Vaccination; Vaccines; Virulence Factors; Work; antigen binding; beta-2 Microglobulin; clinical efficacy; dysbiosis; genomic locus; human subject; in vitro Assay; in vivo; insight; intraperitoneal; memory encoding; monoclonal antibody production; mouse model; neonatal Fc receptor; neutralizing antibody; neutralizing monoclonal antibodies; next generation sequencing; pathogen; premature; programs; receptor binding; recruit; recurrent infection; response; vaccination strategy; vaccine candidate; vaccine development; volunteer

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.

项目摘要/摘要（项目 2） 艰难梭菌疾病复发是一个严重的问题，因为每轮治疗的严重程度都会增加 感染并将局部肠道疾病转变为全身性致命疾病。我们缺乏良好的理解 对感染的免疫反应，可以说是缺乏或错误的，正如反复出现的情况所证明的那样 感染。中和艰难梭菌 (TcdB) 分泌的毒素 B 的血清 IgG 抗体 (Abs) 是最佳相关性 的保护。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 (Bmem) 可以对病原体快速做出反应并产生新的抗体分泌细胞。 然而，在小鼠模型和人类 Bmem 分析中，感染导致的反应占主导地位 尽管一些 IgG+ 和 IgA+ 细胞是明显的，但由 IgM+ 细胞引起。人 TcdB- 的分离和单细胞条形码 特异性 Bmem 随后进行测序和库分析显示 IgG+ 和 IgA+ 细胞已经历 体细胞超突变并具有广泛的可变基因使用，代表了几种独特的 B 细胞克隆。 从选定的 IgG1 基因序列生产单克隆抗体 (mAb) 显示出对 TcdB 的中等亲和力 在一项体外测定中，TcdB 中和效果较差。这项工作引导我们提出一个假设，即 TcdB 特异性 由艰难梭菌诱导的人 B 细胞记忆室编码的 IgG 和 IgA 具有可变性 毒素中和能力。在具体目标 1 中，我们将衡量 TcdB 特定的 Bmem 衍生的影响 单克隆抗体对宿主细胞中毒机制的影响。我们将根据数据库中的基因序列生产单克隆抗体 并招募新的志愿者来扩大 Bmem 曲目的数量。我们将确定 Bmem 的影响- 衍生的单克隆抗体控制宿主细胞中毒的机制，并提供了一个全面的观点 人类 Bmem 细胞编码的 TcdB 特异性抗体在艰难梭菌感染后个体中的功能。具体来说 目标 2：我们将测试 Bmem 细胞衍生的 mAb 抵御活病原体挑战的能力，以及 确定转运至肠道的机制。我们发现新生儿 Fc 受体 (FcRn) 是 将免疫诱导的循环 IgG 递送至肠道并针对艰难梭菌提供保护，而 腹膜内递送至受体小鼠绕过了 FcRn 要求。我们将向腹腔注射单克隆抗体 B6 小鼠以确定人 Bmem 衍生的 mAb 是否具有体内保护作用。我们将使用 FcRn-/- 小鼠表达 人 FcRn 和辅助分子 β2 微球蛋白 (hβ2M) 转基因 (hFcRn:hβ2M ) 以确定 hFcRn 依赖循环 mAb 提供保护。目标 2 将提供有关是否 Bmem 编码的关键数据 单克隆抗体在体内具有保护作用，并决定它们如何到达肠道。项目2具有高度相关性 公共卫生。我们目前对人类受试者对艰难梭菌的体液免疫反应缺乏了解 解释为什么患者容易反复感染的必要机制见解。我们的工作将 确定 Bmem 区室是否不足以编码保护性抗体，并揭示功能性抗体 保护性 Bmem 细胞与非保护性 Bmem 细胞的特性并为疫苗接种策略提供信息。