Polysaccharide Antibody Repertoires

描述（由申请人提供）：本提案代表了我们为了解保护性多糖（PS）抗体（Ab）库的遗传学、细胞基础和体细胞进化所做的持续努力。我们将重点关注肺炎链球菌多糖血清型14（PPS 14）和23 F（PPS 23 F）以及流感嗜血杆菌B型荚膜多糖（HiB PS）的特异性抗体库。初步研究使我们假设原代PPS B细胞库具有低亲和力，并且依赖于由缀合物疫苗接种驱动的超突变，以产生高亲和力的保护性应答。PPS抗体库的分子个体发生将通过从PPS-蛋白结合物疫苗免疫后的婴儿中分离PPS特异性B细胞来检查。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 B片段进行亲和力分析。将选择的Fab片段转化为全长重组Ab以评价调理吞噬活性。这些研究将确定婴儿在应答PPS 14和PPS 23 F时使用的V基因，记录体细胞超突变和/或V区移位在应答成熟过程中发生的程度，并确定该突变的功能后果。我们的体外研究表明，IgH等位基因多态性可以显着影响PS抗体功能。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，以检验以下假设：与使用V3 - 23 * 01等位基因的个体相比，使用V3 - 23*03等位基因的个体将产生更高质量的典型HiB PS Ab和PPS 23 F Ab。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价V基因的使用和突变，并对源自这些克隆的Fab B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合成速率（半衰期），以确定接种是否诱导这些B细胞中的DNA合成（克隆增殖）。这些研究涉及临床相关主题。他们将确定抗体疗效和疾病易感性的遗传决定因素，阐明参与儿科疫苗免疫反应的细胞和过程，并描述对包囊病原体免疫的机制。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.