HUMAN ANTIBODY REPERTOIRE TO H INFLUENZAE B

尽管有抗生素可用，但流感嗜血杆菌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，是其他严重疾病的病原体 会厌炎、化脓性关节炎、蜂窝组织炎等侵袭性疾病 肺炎。Hib被膜多糖(Hib PS)是主要的 毒力因子和针对Hib PS的抗体提供对侵袭性的保护 Hib病。不到两岁的儿童很少或根本不合成。 对Hib PS的抗体和这一组具有最高的 易受感染。这里描述的实验将检验 新生儿和成人Hib PS抗体可变区的差异性 曲目。我们的目标是确定特定的、 生殖系编码的V结构域主导对Hib PS的抗体反应，并且 为了确定免疫能力的年龄依赖性发展是否是 可以用有限的和/或差异的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。分泌Hib-PS特异性单抗的杂交瘤 将从成人外周血单核细胞(MNC)和 来自植入脐带血单个核细胞的高免SCID小鼠。这一小组 将检测人单抗的交叉反应独特型(CRI)的表达 使用针对复发性和非复发性的多克隆和单抗 与Hib PS抗体相关的主要CRI。V区序列 这些单抗将通过对克隆的聚合酶链进行测序来确定 反应扩增，杂交瘤基因。V区序列的比较 CRI的表达有助于阐明CRI的结构相关性 并将描绘出新生儿和成人V的分子多样性 Hib PS的地区剧目。为了进一步本地化CRI决定因素， 将针对合成的多肽制备抗体，这些多肽编码 抗Hib-PS Heavy(H)和Light(L)单抗的保守高变区 锁链。这些抗肽抗体将被检测为独特型 针对人Hib PS特异性单抗及其特异性的研究 孤立的H链和L链。这种方法应该允许识别 编码CRI决定因素的特定高变区，以及 描述H和L链V区在CRI中的单独作用 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‘s 与Hib PS抗体的主要CRI反应将用于 SCID-HU小鼠确定它们是否可以作为替代Hib 疫苗。这些研究应有助于深入了解V区的个体发育 V区结构、独特型与基因表达的关系 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.