HUMAN ANTIBODY REPERTOIRE TO H INFLUENZAE B

尽管有抗生素，流感嗜血杆菌B（HiB） 仍然是一种重要的人类病原体：它是脑膜炎的主要原因 在北美的儿童中，它是其他严重疾病的病原体。 侵袭性疾病如会厌炎、脓毒性关节炎、蜂窝织炎和 肺炎Hib荚膜多糖（Hib PS）是Hib的主要成分。 毒力因子和Hib PS抗体提供了对侵袭性 Hib疾病。两岁以下的儿童合成很少或没有 Hib PS的抗体，这说明该组具有最高的 对感染的易感性这里描述的实验将检查 新生儿和成人Hib PS抗体可变（V）区的多样性 保留曲目。我们的目标是确定在多大程度上， 生殖系编码的V结构域主导对Hib PS的抗体应答，并且 以确定免疫活性的年龄依赖性发展是否 这可以用有限和/或差分V区利用来解释。 将使用独特型和测序分析来描绘V区 表情杂交瘤，分泌Hib-PS特异性单克隆抗体（mAb） 将从成人外周血单核细胞（MNC）制备， 来自移植有脐带血MNC的超免疫scid小鼠。的此面板 将检测人mAb的交叉反应性独特型（CRI）表达 使用多克隆和单克隆抗体， 与Hib PS抗体相关的主要CRI。V区序列 这些单克隆抗体将通过测序克隆，聚合酶链 反应扩增，杂交瘤cDNA。V区序列比较 CRI的表达有助于阐明CRI的结构相关性， 并将描绘新生儿和成人V的分子多样性 Hib PS的区域库。为了进一步定位CRI决定簇， 将制备针对合成肽的抗体 mAb抗Hib PS重链（H）和轻链（L）的保守高变区 店将对这些抗肽抗体进行独特型检测 针对人Hib PS特异性mAb组的特异性及其 分离的H和L链。这一方法应允许确定 编码CRI决定簇的特定高变区，以及 描述了CRI中H和L链V区的各自作用 表情CRI/V区表达与Hib PS的关系 还将检查抗体亲和力和功能活性。MAb的 将给予与Hib PS抗体的主要CRI反应的抗体， scid-hu小鼠，以确定它们是否可以作为Hib的替代物 疫苗这些研究将为深入了解V区的个体发育提供线索 V区结构、独特型与 抗体功能

Despite the availability of antibiotics, Haemophilus influenzae b (Hib) remains a significant human pathogen: it is the leading cause of meningitis in children in North America, and is the etiologic agent in other serious invasive diseases such as epiglottitis, septic arthritis, cellulitis and pneumonia. The Hib capsular polysaccharide (Hib PS) is the primary virulence factor, and antibodies to Hib PS confer protection from invasive Hib disease. Children less than two years of age synthesize little or no antibody to Hib PS and this accounts for this group having the highest susceptibility to infection. The experiments described here will examine the diversity of the neonatal and adult Hib PS antibody variable (V) region repertoire. Our objective is to determine the extent to which particular, germ-line encoded V domains dominate the antibody response to Hib PS, and to determine whether the age-dependent development of immunocompetence, is explicable in terms of limited and/or differential V region utilization. Idiotypic and sequencing analyses will be used to delineate V region expression. Hybridomas, secreting Hib-PS specific monoclonal antibody (mAb) will be prepared from adult peripheral blood mononuclear cells (MNC) and from hyperimmunized scid mice engrafted with cord blood MNC. This panel of human mAb's will be tested for expression of cross-reactive idiotype (CRI) using polyclonal and monoclonal antibodies specific for a recurrent and predominant CRI associated with antibodies to Hib PS. V region sequences of these mAbs will be determined by sequencing cloned, polymerase chain reaction amplified, hybridoma cDNA. Comparison between V region sequence and CRI expression should help elucidate the structural correlates of CRI and will delineate the molecular diversity of the neonatal and adult V region repertoires to Hib PS. To further localize CRI determinants, antibodies will be prepared against synthetic peptides which encode conserved hypervariable regions of mAb anti-Hib PS heavy (H) and light (L) chains. These anti-peptide antibodies will be tested for idiotypic specificity against the panel of human Hib PS-specific mAb's and their isolated H and L chains. This approach should allow for identification of particular hypervariable regions which encode CRI determinants, as well as delineate the individual roles of H and L chain V regions in CRI expression. The relationship between CRI/V region expression and Hib PS antibody affinity and functional activity will also be examined. MAb's reactive with the major CRI of Hib PS antibodies will be administered to scid-hu mice to determine whether they might function as a surrogate Hib vaccine. These studies should provide insight into the ontogeny of V region expression and the relationship between V region structure, idiotypes and antibody function.