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AGING--ANTIBODY RESPONSE TO BACTERIAL AND VIRAL AGS

衰老——对细菌和病毒 AGS 的抗体反应

基本信息

批准号：
6372074
负责人：
Paolo Casali
金额：
$ 34.79万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-08-15 至 2003-07-31
项目状态：
已结题

项目摘要

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.

该提案的总体范围是揭示机制产生针对外源抗原的抗体 (Abs) 的基础（Ags）随着年龄的增长而变化。老年人表现出异常抗体对外源性抗原的反应，特别是对细菌和细菌的反应病毒，包括肺炎链球菌（肺炎球菌）和流感病毒，他们受到显着的影响感染这些和其他疾病后的发病率和死亡率微生物病原体。同样对微生物的异常抗体反应在老年小鼠中发现了 Ag，并与 B 细胞库克隆组成的改变。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的克隆型那些在年轻人中招募来应对相同的外源性 Ags，并且可能反映稳定成分的变化状态 B 细胞库。我们还假设，除了 B细胞克隆型募集的改变，体细胞的机制 B 细胞多样化，即 Ig V(D)J 基因超突变和 Ag的选择是无效的，从而导致不完美老年受试者中 Ag 诱导的抗体的亲和力成熟。这样的无效的体细胞选择机制可能反映出缺陷 B 细胞突变机制所固有的，可能是由根据老年人的记录，有缺陷的 T 细胞会有所帮助，并且会导致对 T 细胞非依赖性以及 T 细胞的异常反应细胞依赖性抗原。为了检验我们的假设，我们建议接种疫苗含老化肺炎球菌多糖及流感病毒疫苗受试者（65 岁及以上），以及作为比较的年轻人成人（20 至 45 岁），并： (i) 分析表型和整个 B 细胞库的克隆型组成，以及它的一些子集的那些，以及表型，频率和细胞前体的克隆型分类产生针对肺炎球菌和流感病毒的 IgM、IgG 和 IgA 抗体银；在最大活化条件和缺乏活化的情况下刺激； (ii) 产生针对肺炎球菌的单克隆抗体 (mAb) 并针对流感病毒 Ag，分析 mAb Ag 结合特性，它们的 VHDJH 和 VLJL 片段的一级结构，以及它们的体细胞点突变的状况；最后，(iii) 验证结构和功能提供的数据对肺炎球菌和流感的选定 B 细胞克隆进行分析病毒，并将其扩展到个体的多个元素克隆型来衡量克隆内多样化的程度组合噬菌体展示中的 Ig 基因“库克隆” 图书馆。抗体反应的细胞和分子特征老年受试者中的肺炎球菌和流感病毒将不仅与年轻人的相应反应相比成人，也适用于天然抗体和广告诱导抗体的人老年受试者对其他微生物抗原的反应，并且可能，因此，帮助设计具体的治疗干预手段。