Modulating affinity maturation during in vivo immunisation

在20世纪，通过接种疫苗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，关于抗体是如何产生的，以及这个过程如何被操纵，仍然有许多基本的生物学问题没有得到解答。例如，(1)我们是否可以通过联合传递抗原特异性抗体来增加针对复杂抗原的抗体的体内亲和力成熟？(2)我们是否可以通过调节B细胞信号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细胞经历多轮突变和选择以逐步改善抗原结合(亲和力成熟)时，就会产生抗体。这一过程在免疫后是自我限制的，并假设有一个亲和力“上限”，超过这个上限对额外的抗体突变没有选择性优势。然而，更高水平的抗体突变(高达45%)是可能的，并在慢性感染期间观察到。因此，该项目旨在建立操纵反应的机制，以产生更多样化的高度突变抗体群体。具体来说，通过回答上述两个问题来检验以下假设：通过联合注射抗原特异性抗体和调节B细胞信号，可以改善对复杂抗原的抗体反应。项目里程碑：(1)将用多亚单位HIV包膜蛋白(Env)免疫三组小鼠，如下所示：(A)对照臂；(B)抗原特异性抗体共传递臂；(C)mTOR抑制剂处理臂。总共，每组将每两周接受五次佐剂免疫原剂量。(2)用单细胞流式细胞术分离免疫原特异性的单抗，可以在单个克隆水平上评估亲和力成熟程度。(3)通过对大量B细胞群体的NGS分析，将评估整个保存库的亲和力成熟度和克隆多样性。时间表：学生将在UCL的前6个月生产免疫试剂，然后他们将在GSK花8个月的时间处理免疫后样本，生成NGS数据。然后，他们将返回伦敦大学学院分离单抗，并优化分析NGS数据的方法。相关性：该项目中产生的知识对于疫苗和单抗的开发都将是重要的。因此，这项建议与BBSRC的两个战略研究优先事项保持一致：工业生物技术和生物能源与生物科学促进健康。它将利用关键的研究机会，发展基础生物科学，作为开发生物制品(被确定为工业研究合作目标的四项关键产出之一)的基础。这项工作还将解决挑战领域(BBSRC生物科学促进健康战略框架)，提供对基本生物学机制的新理解，并利用大数据技术开发创新的研究工具，即NGS。合作伙伴关系：UCL团队拥有分析免疫后血清样本和单抗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，以及全面运作的III级遏制实验室。葛兰素史克的体内选择团队在对小鼠进行体内免疫方面拥有广泛的专业知识，并已开发出方法学(与麦考伊博士借调到葛兰素史克)，以生成用于Illumina NGS分析的抗体库。

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.