Modulating affinity maturation during in vivo immunisation

体内免疫过程中调节亲和力成熟

• 批准号: 2243420
• 金额: --
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2243420
• 关键词: Modulating; affinity; maturation; during; vivo

During the 20th century, the ability to induce antibody responses via vaccination had a huge impact on livestock husbandry and human life expectancy. However, there remain many unanswered basic biological questions about how antibodies are generated and how this process can be manipulated. For example, (1) Can we increase in vivo affinity maturation of antibodies against complex antigens by co-delivery of antigen-specific antibody? (2) Can we increase diversity of the antibody response against complex antigens by modulating B cell signalling? Antibodies are produced when antigen-specific B cells undergo multiple rounds of mutation and selection for progressively improved antigen binding (affinity maturation). This process is self-limiting following immunisation and there is postulated to be an affinity "ceiling" above which there is no selective advantage for additional antibody mutations. However, higher levels of antibody mutation (up to 45%) are possible and have been observed during chronic infection. Therefore, this project aims to establish mechanisms to manipulate the response to produce a more diverse population of highly mutated antibodies. Specifically, by addressing the two questions above to test the following hypothesis: the antibody response to complex antigens can be improved by co-administration of antigen-specific antibodies and modulation of B cell signalling.Project milestones: (1) Three groups of ten mice will be immunised with the multi-subunit HIV envelope protein (Env) as follows: (A) control arm; (B) Antigen-specific antibody co-delivery arm; (C) mTor inhibitor treated arm. In total, each group will receive five adjuvanted immunogen doses at two-week intervals. (2) Single cell FACS to isolate immunogen-specific monoclonal antibodies (mAbs) will allow assessment of affinity maturation at the level of individual clones. (3) Affinity maturation across the repertoire and also clonal diversity will be evaluated by NGS analysis of bulk B cell populations. Timeline: The student will spend the first 6 months at UCL producing immunisation reagents, they will then spend 8 months at GSK to process post-immunisation samples, generate NGS data. They will then return to UCL to isolate mAbs and optimise methods to analyse the NGS data. Relevance: The knowledge generated in this project will be important both for development of vaccines and mAbs. Thus, this proposal is aligned with two of the BBSRC's strategic research priorities: Industrial Biotechnology and Bioenergy and Bioscience for health. It will take advantage of key research opportunities to develop basic bioscience underlying the development of biologics (one of four key outputs identified as targets for industrial research collaborations). This work will also address the challenge areas (BBSRC Bioscience for Health Strategic Framework) by providing new understanding of fundamental biological mechanisms and develop innovative research tools using big data technology, namely NGS. Partnership: The UCL team have all the skills and equipment required to analyse both the post immune serum samples and mAbs, including access to extensive bioinformatics infrastructure, and a fully operational containment level III laboratory. The in vivo selections team at GSK has extensive expertise in performing in vivo immunisations of mice and have developed methodology (with Dr McCoy on secondment at GSK) to generate antibody repertoire libraries for Illumina NGS analysis.

在20世纪，通过疫苗接种诱导抗体反应的能力对畜牧业和人类预期寿命产生了巨大影响。然而，关于抗体如何产生以及如何控制这一过程，仍有许多未解的基本生物学问题。例如，(1)我们能否通过共递送抗原特异性抗体来提高针对复杂抗原的抗体在体内的亲和力成熟？(2)我们能否通过调节B细胞信号传导来增加针对复杂抗原的抗体反应的多样性？当抗原特异性B细胞经过多轮突变和选择以逐渐改善抗原结合（亲和成熟）时，产生抗体。这一过程在免疫后是自我限制的，并且假定存在一个亲和力“上限”，在此上限之上，对于额外的抗体突变没有选择优势。然而，在慢性感染期间可能出现更高水平的抗体突变（高达45%）。因此，本项目旨在建立机制来操纵反应，以产生更多样化的高度突变抗体群体。具体来说，通过解决上述两个问题来验证以下假设：抗体对复杂抗原的反应可以通过抗原特异性抗体的联合施用和B细胞信号传导的调节来改善。项目里程碑：(1)用多亚单位HIV包膜蛋白（Env）免疫三组10只小鼠，如下：(A)对照组；(B)抗原特异性抗体共递送臂；(C) mTor抑制剂治疗组。总的来说，每组将每隔两周接受五剂免疫原佐剂。(2)用于分离免疫原特异性单克隆抗体（mab）的单细胞FACS将允许在单个克隆水平上评估亲和成熟。(3)通过对大量B细胞群体的NGS分析来评估整个库的亲和成熟度和克隆多样性。时间：学生将在伦敦大学学院花6个月时间生产免疫试剂，然后在GSK花8个月时间处理免疫后样品，生成NGS数据。然后，他们将返回伦敦大学学院分离单克隆抗体，并优化分析NGS数据的方法。相关性：本项目产生的知识对疫苗和单克隆抗体的开发都很重要。因此，该提案与BBSRC的两个战略研究重点：工业生物技术和生物能源以及健康生物科学是一致的。它将利用关键的研究机会来发展生物制剂开发的基础生物科学（确定为工业研究合作目标的四个关键产出之一）。这项工作还将通过提供对基本生物学机制的新理解和开发利用大数据技术（即NGS）的创新研究工具，解决挑战领域（BBSRC生物科学促进健康战略框架）。伙伴关系：伦敦大学学院的团队拥有分析免疫后血清样本和单克隆抗体所需的所有技能和设备，包括获得广泛的生物信息学基础设施，以及一个全面运作的III级控制实验室。GSK的体内选择团队在进行小鼠体内免疫方面拥有丰富的专业知识，并开发了方法（与GSK借调的McCoy博士一起）来生成用于Illumina NGS分析的抗体库库。