Polysaccharide Antibody Repertoires

描述(由申请人提供)：本提案代表我们继续努力了解保护性多糖(PS)抗体(Ab)谱系的遗传学、细胞学基础和体细胞进化。我们将集中于肺炎链球菌多糖14型(PPS 14)和23F(PPS 23F)以及b型流感嗜血杆菌荚膜多糖(Hib PS)的特异性抗体谱系。初步研究使我们假设，初级PPS B细胞谱系是低亲和力的，依赖于结合疫苗驱动的超突变来产生高亲和力的保护性反应。在PPS蛋白结合疫苗免疫后，将通过从婴儿中分离PPS特异性B细胞来检查PPS抗体谱系的分子个体发育。将对单个PPS特异性B细胞或其克隆产物使用的可变区(V)区基因进行测序，并对单克隆性Fab片段进行亲和力分析。选定的Fab片段将被转化为全长重组抗体，以评估吞噬细胞的活性。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过度突变和/或V区转移的程度，并确定这种突变的功能后果。我们的体外研究表明，IgH等位基因的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，以检验这样的假设，即使用V3-23*03等位基因的个体将产生比使用V3-23*01等位基因的个体更高质量的典型Hib PS抗体和PPS 23F抗体。关于外周血中IgM记忆B细胞的特异谱及其在PS免疫中的作用，我们知之甚少。我们将确定从接种疫苗的成人中分离出的PS特异性IgM‘记忆’B细胞的V区谱系。V基因的使用和突变将在单个IgM记忆B细胞或扩展克隆中进行评估，PS结合研究将在来自这些克隆的Fab片段上进行。我们将使用一种稳定的活体同位素标记方法来测定免疫前后IgM记忆性B细胞和其他血液B细胞亚群的DNA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(克隆性增殖)。这些研究涉及临床相关主题。他们将确定抗体效力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，并描述对被包裹的病原体免疫的潜在机制。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.