AGING--ANTIBODY RESPONSE TO BACTERIAL AND VIRAL AGS

这项提案的总体范围是揭示 外源抗原抗体(Abs)的产生 (AGS)，因为它们随着年龄的变化而变化。老年人出现抗体异常 对外源AGS的反应，特别是对细菌和 病毒，包括肺炎链球菌(肺炎球菌)和 流感病毒，他们受影响的比率很高 感染这些病毒和其他病毒后的发病率和死亡率 微生物病原体。对微生物的类似异常抗体反应 已在老年小鼠中发现AGS，并与 B细胞谱系克隆组成的变化。我们 假设老年人对微生物的异常反应 病原体是由于招募了不同于 那些在年轻人中招募的人对同样的外源性 Ags，并可反映稳定剂组成的变化- 国家B细胞保留曲目。我们还假设，除了 一种改变的B细胞克隆性募集，体细胞机制 B细胞多样化，即Ig V(D)J基因过度突变和 由银选择，是无效的，从而导致不完美 老年人Ag诱导抗体亲和力成熟的研究是这样的 无效的体细胞选择机制可能反映了一种缺陷 B细胞突变机制所固有的，可能与 T细胞缺陷是有帮助的，正如在老年人中记录的那样，并且会 导致对T细胞非依赖性以及T细胞的异常反应 依赖细胞的AGS。为了检验我们的假设，我们建议接种疫苗。 肺炎球菌多糖和流感病毒疫苗老化 受试者(65岁及以上)和年轻人(作为比较) 成年人(20至45岁)，并：(I)分析表型 和作为一个整体的B细胞库的克隆型组成，以及 它的一些子集，以及表型， 频率和细胞前体的克隆型分类 产生抗肺炎球菌和流感病毒的IgM、IgG和IgA抗体 Ags；在最大激活条件下和不激活情况下 刺激；(Ii)产生抗肺炎球菌的单抗 对于流感病毒AGS，分析mAbAg结合特性， 它们的VHDJH和VLJL片段的一级结构，以及它们的 关于体细胞点突变的地位；以及，最后，(3) 验证由结构和功能提供的数据 肺炎球菌和流感病毒B细胞克隆的筛选分析 病毒，并将其扩展到个人的多个元素 克隆类型用来衡量克隆内多样化的程度 免疫球蛋白基因在噬菌体组合展示中的“谱系克隆” 图书馆。抗体反应的细胞和分子特征 对肺炎球菌和流感病毒的老年受试者 不仅与年轻人的相应反应相比较 成虫，也对那些自然和Ad诱导的抗体 老年人对其他微生物AGS的反应，并可能， 因此，帮助设计治疗干预的具体手段。

The overall scope of this proposal is to uncover the mechanisms underlying the generation of antibodies (Abs) to exogenous antigens (Ags) as they change with aging. Aged people display abnormal Ab responses to exogenous Ags, particularly those on bacteria and viruses, including Streptococcus pneumoniae (Pneumococcus) and influenza virus, and they are affected with significant rates of morbidity and mortality following infection with these and other microbial pathogens. Similarly abnormal Ab responses to microbial Ags have been found in aged mice and have been related to alterations of the clonal composition of the B cell repertoire. We hypothesize that in aged humans the-abnormal responses to microbial pathogens are due to the recruitment of clonotypes different from those recruited in young adults in response to the same exogenous Ags, and may reflect alterations of the composition of the steady- state B cell repertoire. We also hypothesize that, in addition to an altered B cell clonotypic recruitment, the mechanisms of somatic B cell diversification, i.e., Ig V(D)J gene hypermutation and selection by Ag, are ineffective, thereby leading to imperfect affinity maturation of Ag-induced Abs in aged subjects. Such ineffective somatic selection mechanisms may reflect a defect inherent to the B cell mutational machinery, perhaps compounded by a defective T cell help, as documented in the elderly, and would result in abnormal responses to T cell-independent as well as T cell-dependent Ags. To test our hypotheses, we propose to vaccinate with Pneumococcus polysaccharide and influenza virus vaccines aged subjects (65 years of age and older) and, for comparison, young adults (20 to 45 years of age), and to: (i) analyze the phenotypic and clonotypic composition of the B cell repertoire as a whole, and those of some of its subsets, as well as the phenotype, the frequency, and the clonotypic assortment of the precursors of cells producing IgM, IgG, and IgA Abs to Pneumococcus and influenza virus Ags; under maximal activating conditions and absence of activating stimuli; (ii) generate monoclonal antibodies (mAbs) to Pneumococcus and to influenza virus Ags, analyze the mAb Ag-binding properties, the primary structure of their VHDJH and VLJL segments, and their status with respect to somatic point-mutations; and, finally, (iii) validate the data provided by the structural and functional analyses of selected B cell clones to Pneumococcus and influenza virus, and extend them to multiple elements of individual clonotypes to measure the extent of intraclonal diversification by Ig gene "repertoire cloning" in combinatorial phage display libraries. The cellular and molecular features of the Ab response to Pneumococcus and influenza virus in aged subjects will be compared not only to those of the corresponding responses in young adults, but also to those of the natural and Ad-induced Ab responses to other microbial Ags in aged subjects, and may, therefore, help design specific means of therapeutic intervention.