Polysaccharide Antibody Repertoires

描述（由申请人提供）：该提案代表了我们在了解保护性多糖（PS）抗体（Ab）库的遗传学，细胞基础和体细胞进化方面的持续努力。我们将重点关注肺炎链球菌多糖血清型14 （PPS 14）和23F （PPS 23F）以及流感嗜血杆菌b型荚膜多糖（Hib PS）特异性的Ab谱。初步的研究使我们假设原代PPS B细胞库具有低亲和力，并依赖于偶联疫苗驱动的高突变，以产生高亲和力的保护性反应。通过从接种PPS蛋白结合疫苗后的婴儿中分离PPS特异性B细胞，来检测PPS抗体库的分子个体发生。将对单个pps特异性B细胞或其克隆产物使用的可变(V)区基因进行测序，并对单克隆Fab片段进行亲和分析。选定的Fab片段将转化为全长重组抗体，以评估调理自噬细胞活性。这些研究将确定婴儿在PPS 14和PPS 23F反应中使用的V基因，记录在反应成熟过程中发生体细胞超突变和/或V区移位的程度，并确定这种突变的功能后果。我们的体外研究表明，IgH等位基因多态性可以显著影响PS Ab的功能。因此，我们将在婴儿和成人中进行疫苗接种和B细胞克隆研究，以验证使用V3-23*03等位基因的个体比使用V3-23*01等位基因的个体产生更高质量的标准Hib PS抗体和PPS 23F抗体的假设。关于外周血IgM记忆B细胞的特异性库或它们在PS免疫中的作用知之甚少。我们将鉴定从接种疫苗的成人中分离的ps特异性IgM“记忆”B细胞的V区库。将在单个IgM记忆B细胞或扩增克隆中评估V基因的使用和突变，并对来自这些克隆的Fab片段进行PS结合研究。我们将使用稳定同位素体内标记方法测定IgM记忆B细胞和其他血液B细胞亚群在接种前后的DMA合成率（半衰期），以确定疫苗接种是否诱导这些B细胞的DNA合成（克隆增殖）。这些研究涉及临床相关的主题。他们将确定Ab有效性和疾病易感性的遗传决定因素，阐明参与儿童疫苗免疫反应的细胞和过程，并描述对封装病原体的免疫机制。这项研究将加深我们对人类免疫系统的理解，并可能有助于设计更好的疫苗。

DESCRIPTION (provided by applicant): This proposal represents our continuing efforts to understand the genetics, cellular basis and somatic evolution of protective polysaccharide (PS) antibody (Ab) repertoires. We will focus upon Ab repertoires specific for Streptococcus pneumoniae polysaccharide serotypes 14 (PPS 14) and 23F (PPS 23F) and the Haemophilus influenzae type b capsular polysaccharide (Hib PS). Preliminary studies lead us to hypothesize that the primary PPS B cell repertoire is of low affinity and relies upon hypermutation, driven by conjugate vaccination, to generate a high affinity, protective response. The molecular ontogeny of the PPS Ab repertoire will be examined by isolating PPS-specific B cells from infants following immunization with PPS-protein conjugate vaccines. The variable (V) region genes used by individual PPS-specific B cells or their clonal products will be sequenced and affinity analyses will be performed on monoclonal Fab fragments. Selected Fab fragments will be converted to full-length recombinant Abs in order to evaluate opsonophagocytic activity. These studies will identify the V genes used by infants in response to PPS 14 and PPS 23F, document the extent to which somatic hypermutation and/or V region shifts occur in the maturation of the response and determine the functional consequences of this mutation. Our in vitro studies indicate that IgH allelic polymorphisms can dramatically influence PS Ab function. Accordingly, we will perform vaccination and B cell cloning studies in infants and adults to test the hypothesis that individuals utilizing the V3-23*03 allele will produce higher quality canonical Hib PS Abs and PPS 23F Abs than individuals using the V3-23*01 allele. Little is known about the specificity repertoire of peripheral blood IgM memory B cells or their role in PS immunity. We will identify the V region repertoire of PS-specific IgM 'memory' B cells isolated from vaccinated adults. V gene usage and mutation will be evaluated in individual IgM memory B cells or expanded clones, and PS binding studies will performed on Fab fragments derived from these clones. We will use a stable isotope in vivo labeling method to determine the DMA synthetic rates (half lives) of IgM memory B cells and other blood B cell subsets both before and after vaccination to determine whether vaccination induces DNA synthesis (clonal proliferation) in these B cells. These studies address clinically relevant topics. They will identify genetic determinants of Ab efficacy and disease susceptibility, elucidate the cells and processes involved in the immune response to pediatric vaccines, and delineate the mechanisms underlying immunity to encapsulated pathogens. This research will deepen our understanding of the human immune system and may contribute toward the design of better vaccines.