Regulation of the Immune Response to Polysaccharides and Polysaccharide Conjuga

多糖类抗原的免疫应答受高度调控，具有亚类受限、基因使用可变区、特异性好和亲和力强等特点。未与蛋白质结合的简单PS(如细菌Levan，脑膜炎奈瑟氏菌C群(MCPS))可引起胸腺非依赖性(TI)反应。另一方面，PS与蛋白质(如与破伤风类毒素偶联的MCPS-TT)结合后，会引起一种不同类型的反应，称为胸腺依赖(TD)。我们早些时候对抗BL抗体的分析表明，对菊糖(In)分支决定簇的初步反应是胚系的。然而，两次注射BL可诱导出具有体细胞突变和较高亲和力的IgM单抗。我们现在已经对生殖系抗体进行了定点突变，并确定了导致亲和力较高或较低的特定位点。数据表明芳香族氨基酸在抗PS抗体CDRs中的重要性。在我们对抗In反应中多样性调节的分析中，我们将SR1多样性基因定位在小鼠14号染色体上。最近在TCR KO小鼠中的研究表明，对In的反应不仅是TI，而且当加回时不受T细胞的影响。以往对抗MCPS和抗MCPS TT单抗的分析表明，VH基因家族的使用主要由VHJ558控制。对抗MCPS单抗的序列分析表明，在这种反应中使用了几个不同的胚系基因，即使是具有相同良好特异性的单抗也是如此。一种特殊的VH：VL Pait，VH3609/Vk23，与天然MCPS而不是非O-乙酰化MCPS具有良好的反应性特异性。对MCPS的反应很少看到体细胞突变，但当观察到时，与亲和力的增加相关。我们实验室早期的小鼠模型研究表明，即使作为TD-MCPS-TT结合物给药，对MCPS的免疫反应也有发育延迟。因此，研究人员对新生小鼠TD结合物反应延迟是否与抗原提呈缺陷有关进行了研究。我们发现，成人B细胞比全脾细胞或巨噬细胞更有效地向T细胞递呈TT或MCPS-TT，并且成体树突状细胞是该系统中最有效的抗原提呈细胞，其效率约为B细胞的2倍。值得注意的是，新生儿B细胞和新生儿树突状细胞呈递抗原的能力均有缺陷。最近的研究表明，B细胞和DC上的谱系特异性标志物都有发育增加，B细胞上MHC II类和DC上CD11c的增加与其功能的发展有关。这些数据表明，提高新生儿细胞呈递抗原能力的因素可能有助于刺激新生儿对结合疫苗的反应。儿童疫苗倡议的目标之一是减少为儿童充分接种疫苗所需的接触人数。为了实现这一目标，已经对几种组合疫苗进行了安全性和婴儿免疫原性测试。在智利进行的初步研究表明，当婴儿在同一注射器中或单独注射时，对流感嗜血杆菌(Hib)多糖(PRP；多聚核糖基核糖核糖磷脂)以及百日咳疫苗与Hib结合疫苗(PRP-T)联合接种的百日咳成分的抗体应答降低。我们已经开发了一个模型，用于研究联合疫苗对单个成分的反应的影响。我们观察到DTaP干扰了对Hib-TT的抗Hib反应，我们将在来年研究这种干扰的基础。我们还观察到，Hib-TT干扰了对所有三种类型的脊髓灰质炎的反应，给出了IPV。这方面的基础也将在来年进行审查。

The immune response to polysaccharide (PS) antigens is highly regulated and has several distinguishing features including restricted subclass, variable region gene usage, fine specificity, and avidity. Simple PS not conjugated to protein (such as bacterial Levan (BL, Neisseria meningitidis group C (MCPS)) elicit a thymus-independent (TI) response. PS conjugated to proteins (such as MCPS coupled to tetanus toxoid, (MCPS- TT)), on the other hand, elicit a different type of response, termed thymus-dependent (TD). Our earlier analysis of anti-BL antibodies had shown a germline primary response to the inulin (In) branch determinants. Two injections of BL, however, elicited IgM mAb with somatic mutations and higher affinity. We have now performed site-directed mutagenesis of the germline antibodies and identified specific sites resulting in higher or lower affinity. Data show the importance of aromatic amino acids in the CDRs of anti-PS antibodies. In our analysis of the regulation of diversity in the anti-In response we have mapped the Sr1 diversity gene to mouse chromosome 14. Recent studies in TCR KO mice have shown that the response to In is not only TI, but is not influenced by T cells when added back. Previous analyses of anti-MCPS and anti-MCPS TT mAb reveal that VH gene family usage is dominated by VHJ558. Sequence analysis of anti MCPS mAb shaows that several different germline genes are used in this response, even by mAb of the same fine specificity. One particular VH:VL pait, VH3609/Vk23, correlates with a fine specificity of reactivity with native MCPS and not non-O-acetylated MCPS. Few somatic mutations are seen in response to MCPS, but when observed, correlate with an increase in affinity. Earlier mouse model studies in our laboratory indicated a developmental delay in the immune response to MCPS even when it was administered as TD MCPS-TT conjugates. Therefore, studies were undertaken to examine whether the delay in response TD conjugates in neonatal mice is due to defective antigen presentation. We have found that adult B cells were much more effective in presenting TT or MCPS-TT to T cells than total spleen cells or macrophages and that adult dendritic cells were the most effective antigen presenting cells in this system,the efficiency being about 2-fold higher than B cells. Of significance, both neonatal B cells and neonatal dendritic cells were defective in their ability to present antigen. Recent studies demonstrate that there is a developmental increase in lineage -specific markers on both B cells and DC and that the increase in MHC class II on B cells and CD11C on DC correlates with the development of their functional capacities. The data suggest that factors that inprove the ability of neonatal cells to present antigen might be useful in stimulating neonatal responses to conjugate vaccines. One of the goals of the Children's Vaccine Initiative is to reduce the number of contacts required to immunize a child fully. To meet this goal several combined vaccines have been tested for their safety and immunogenicity in infants. Initial studies conducted in Chile showed a decrease in antibody response to Haemophilus influenzae b (Hib) polysaccharide (PRP; polyribosylribitolphosphate)and to the pertussis component of a DTP vaccine combined with an Hib conjugate (PRP-T) vaccine when the preparation was administered to infants in the same syringe or separately. We have developed a model in SW outbred mice to examine the effects of combining vaccines on the response to individual components. We have observed that DTaP interferes with the anti-Hib response to Hib- TT and we will examine the basis for this interference in the coming year. We also observed that Hib-TT interfered with the response to all three types of polio, given as IPV. The basis for this will also be examined in the coming year.