Engineering of B cell targeted antigens and pathogens

描述(由申请人提供)：为了产生长寿命、高亲和力的抗体反应，B细胞需要从抗原特异的CD4T细胞获得同源帮助。B细胞招募帮助的能力取决于它们将抗原或病原体内化、降解并通过其MHC II类蛋白呈递衍生多肽的能力。这项提议的核心是这样一个问题，即需要CD4T细胞的B细胞是否比最初驱动CD4T细胞扩张的树突状细胞(DC)帮助展示不同的抗原衍生多肽亚群。我们假设，B细胞和DC展示的病原体和抗原衍生多肽的谱系将是不同的，B细胞呈递的多肽只是DC展示的多肽的一个子集。这种由B细胞呈现的不完整的表位模式将导致CD4T细胞的参与受到限制，从而有助于产生高亲和力抗体。限制我们解决这一重要问题的主要因素是，任何特定抗原的抗原特异性B细胞在体内的出现频率都非常低。这些异常低的频率阻碍了对B细胞的跟踪和在正常生理条件下对其多肽显示的评估。我们建议通过利用B细胞受体(BCR)转基因小鼠来解决这个问题，在BCR转基因小鼠中，大多数B细胞在其细胞表面表达一种已知的、定义的免疫球蛋白。这项提议中的实验的目的是利用分子工程赋予抗原蛋白或病毒被bcr转基因小鼠的免疫球蛋白受体特异性识别的能力。我们将使用噬菌体展示文库来鉴定选择性地与所定义的单克隆免疫球蛋白分子结合的多肽模拟物(模拟表位)，然后将这些模拟表位转移到几个选定的抗原上，包括甲型流感病毒。这一策略将允许有选择地将抗原或病毒靶向抗原特异性免疫球蛋白受体转基因B细胞。这些独特试剂的成功衍生将使我们能够评估免疫球蛋白介导的B细胞摄取病毒或抗原后CD4T细胞表位的产生。最重要的是，使用衍生的抗原和B细胞受体转基因小鼠将大大提高我们了解流感病毒与抗原特异性B细胞之间的相互作用以及B细胞与CD4T细胞之间的同源相互作用的能力。 与公共卫生相关：为了使疫苗成功地激发对病原体挑战的保护性抗体反应，B淋巴细胞需要能够从另一种称为T细胞的淋巴细胞系中获得“帮助”。我们的实验旨在产生几个关键的实验工具，使我们能够解剖并最终控制T细胞的焦点，使它们能够更有效地为产生抗体的细胞提供帮助。通过这些实验，我们将获得提高疫苗效力所需的洞察力。

DESCRIPTION (provided by applicant): To generate a long-lived, high-affinity antibody response, B-cells need to obtain cognate help from antigen-specific CD4+ T cells. The ability of B cells to recruit help depends on their ability to internalize the antigen or pathogen, degrade it, and present the derived peptides via their MHC class II proteins. At the core of this proposal is the question of whether B cells in need of CD4 T cell help display a distinct subset of antigen-derived peptides than do the dendritic cells (DC) that initially drive CD4 T cell expansion. We hypothesize that the repertoire of pathogen and antigen-derived peptides displayed by B cells and DC will be distinct and that B cell-presented peptides will be only a subset of that displayed by DC. This incomplete pattern of epitope presentation by B cells will lead to limitations on the participation of CD4 T cells to deliver help for production of high affinity antibodies. The main element that has limited our ability to address this important issue is that antigen-specific B cells for any given antigen are present in exceeding low frequencies in vivo. These exceptionally low frequencies prevent tracking of the B cells and evaluation of their peptide display under normal physiological conditions. We propose to solve this problem by taking advantage of B cell receptor (BcR) transgenic mice where a majority of the B cells express a known, defined immunoglobulin on their cell surface. The goal of the experiments in this proposal is to use molecular engineering to endow an antigenic protein or virus the ability to be specifically recognized by the immunoglobulin receptor of BcR transgenic mice. We will use phage display libraries to identify peptide mimics (mimetopes) that bind selectively to the defined monoclonal immunoglobulin molecule and then transfer these mimetopes to several selected antigens, including influenza A virus. This strategy will allow selective targeting of the antigen or virus to the antigen-specific immunoglobulin receptor transgenic B cells. Successful derivation of these unique reagents will allow us to assess CD4 T cell epitope generation after immunoglobulin-mediated uptake of virus or antigen by B cells. Most importantly, use of the derived antigens and the B cell receptor transgenic mice will greatly enhance our ability to understand the interaction between influenza virus and antigen-specific B cells and cognate interactions between B cells and CD4 T cells in vivo. PUBLIC HEALTH RELEVANCE: For vaccination to be successful in eliciting a protective antibody response to pathogen challenge, B lymphocytes need to be able to recruit "help" from another lineage of lymphocytes called T cells. Our experiments are aimed at generating several key experimental tools that will allow us to dissect and ultimately control the focus of T cells, to allow them to more efficiently provide help to antibody producing cells. Through these experiments, we will gain the insight needed to improve vaccine efficacy.