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） 梭状芽胞杆菌艰难疾病复发是一个严重的问题，因为每一轮的严重程度都会增加 感染并将区域性促进疾病转化为系统性致命疾病。我们缺乏良好的理解 对感染的免疫反应，可以说是缺乏或错过的，这是反复出现的 感染。艰难梭菌（TCDB）中和毒素B的血清IgG抗体（ABS）是最好的相关性 保护。理想情况下，感染将刺激原发性毒素中和AB反应以及影响毒素 特定的记忆B细胞（BMEM）可以快速响应病原体并产生新的AB分泌细胞。 但是，在小鼠模型和人类BMEM的分析中，感染导致反应主导 通过IgM+细胞，尽管某些IgG+和IgA+细胞得到了证明。人类TCDB-的分离和单细胞条形码 特定的BMEM然后进行测序和曲目分析表明，IgG+和IgA+细胞已经发生了 体细胞超突变，具有可变基因使用的广度，代表了几个独特的B细胞克隆。 从选定的IgG1基因序列中产生单克隆ABS（mAb）揭示了对TCDB的现代亲和力 在一个体外测定中，TCDB神经化差。这项工作指导了我们一个假设，即TCDB特定于 艰难梭菌诱导的人B细胞记忆室编码的IgG和IgA具有可变 毒素中和化的能力。在特定目标1中，我们将衡量TCDB特异性BMEM衍生的影响 mAb关于宿主细胞中毒的机制。我们将从数据库中的基因序列产生mAb 并招募新的志愿者来扩大BMEM替代的数量。我们将确定BMEM-的影响 在控制宿主细胞中毒的机制上衍生的mAb，并提供了全面的视图 艰难梭菌感染后，人BMEM细胞编码的TCDB特异性ABS的功能。具体 AIM 2：我们将测试BMEM细胞衍生的MAB防止现场病原体挑战和 确定向肠道运输的机理。我们证明了新生儿FC受体（FCRN）需要 将免疫引起的循环IgG传递到肠道并防止艰难梭菌进行保护，而 腹膜内递送到接受者小鼠绕过了FCRN要求。我们将把腹膜内mab传递给 B6小鼠确定人体BMEM衍生的mAb是否在体内受到保护。我们将使用表达的fcrn - / - 鼠标 人FCRN和配件分子β2微球蛋白（Hβ2M）翻译（HFCRN：Hβ2M）确定 HFCRN依赖性通过循环mAb来保护。 AIM 2将提供有关BMEM是否编码的关键数据 mAb在体内受到保护，并确定它们如何到达肠道。项目2与 公共卫生。我们目前对人类受试者中对艰难梭菌的体液免疫反应的理解缺乏 解释为什么患者容易复发感染的必要机械见解。我们的工作将 确定BMEM室是否不充分编码保护性ABS并揭示功能 受保护与非保护BMEM细胞的特性，并为疫苗接种策略提供信息。