Mechanisms of differential responses to whole cell and acellular pertussis vaccination

项目总结 项目2 在20世纪90年代中期，疫苗相关的副作用促使整个百日咳(WP)疫苗被替换为 新的无细胞百日咳疫苗(AP)。出乎意料的是，最近百日咳病例有所增加， 尤其是在青少年中。在初步数据中，我们比较了1995年前出生的人和1995年接种疫苗的人 患有湿性肺炎的婴儿，出生于1996年或更晚的人，在婴儿时期接种了AP疫苗。激活诱导 标记物(AIM)分析允许直接检查体外反应，突出Th2与Th1/Th17 百日咳(PT)特异性CD4+T细胞的极化。值得注意的是，我们发现了对 当代AP助推器，尽管第一次AP或WP启动发生在18年前。AP 启动与1)PT反应中IL-9和转化生长因子-β的增加以及IL-17的产生减少有关 体外表位，以及2)体外扩增记忆细胞的能力有缺陷。 AP免疫和对PT表位的体外增殖反应。然而，它的生物学意义 这些观察受到以下事实的限制：只有血液中的反应才被测量，以及是否相同 在记忆T细胞驻留和免疫的淋巴组织中，原位观察到表型差异 回应是经过精心策划的。因此，我们建议定义潜在的分子机制 原始AP和WP供体IL-9/IL-17极化的差异(目标1)。我们将定义细胞 AP疫苗免疫后IL-9+PT特异性CD4+T细胞的特性及其分子机制 发展。我们将通过有针对性的流动研究在WP疫苗启动的个体中看到的IL-17签名 流式细胞术和AIM研究，并对PT特异性的CD4+T细胞进行单细胞RNA-SEQ。 在目标2中，涉及差异增殖的机制将通过在体外阻断转化生长因子-b来解决。 相关途径，以及下调ANAPC2和WDR1基因，这在初步鉴定 数据。最初以AP和WP启动的供者差异增殖的细胞基础将是 通过细胞耗尽实验进行研究。 AIM 3将使用淋巴结部(LN)细针抽吸物(FNA)来询问个体的PT反应 最初用WP或AP启动以评估不同的生发中心(GC)TFH细胞和GC B细胞反应 在疫苗接种者的LN中，定义WP-VS AP-PRI的差异抗体同型反应 个体由于GC、TfH辅助功能对B细胞的偏倚，并确定循环中的CD4+T细胞是否相关 随着疫苗免疫反应的引流，LN是免疫应答的主要部位。总之，我们 相信这项研究的结果将阐明AP疗效较低的分子原因，并说明人类 CD4+T细胞生物学原理一般适用于疫苗学和免疫学。

PROJECT SUMMARY Project 2 In the mid-1990s, vaccine-related side effects prompted the replacement of the whole Pertussis (wP) vaccine by a new acellular Pertussis vaccine (aP). Unexpectedly, whooping cough cases have recently increased, particularly in teenagers. In the preliminary data, we compared individuals born before 1995 and vaccinated in infancy with wP, with individuals born in 1996 or later and vaccinated with aP in infancy. Activation Induced Marker (AIM) assays allowed examination of responses directly ex vivo, highlighting a Th2 vs Th1/Th17 polarization of pertussis (PT)-specific CD4+ T cells. Remarkably, we detected differences in response to a contemporary aP booster, even though the first aP or wP priming occurred more than 18 years previously. aP priming was associated with 1) increased IL-9 and TGF-β and decreased IL-17 production in response to PT epitopes ex vivo, and 2) defective ex vivo capacity to expand memory cells one to two months following a booster aP immunization and in vitro proliferation in response to PT epitopes. However, the biological significance of these observations is limited by the fact that only responses in blood are measured and whether the same phenotypic differences are observed in situ, in the lymphatic tissues where memory T cell reside and immune responses are orchestrated. Accordingly, we propose to define the molecular mechanisms underlying the differential IL-9/IL-17 polarization of donors originally primed with aP vs wP (Aim 1). We will define the cellular characteristics of IL-9+ PT-specific CD4+ T cells after aP vaccination and molecular mechanisms underlying their development. We will study the IL-17 signature seen in wP vaccine-primed individuals, by targeted flow cytometry and AIM studies, and perform single-cell RNA-seq with the PT-specific CD4+ T cells. In Aim 2, the mechanisms involved in differential proliferation will be addressed by blocking in vitro TGF-b related pathways, and downregulating the ANAPC2 and WDR1 genes, which were identified in the preliminary data. The cellular basis of the differential proliferation of donors originally primed with aP vs wP will be investigated by cell depletion experiments. Aim 3 will use Lymph Node (LN) Fine Needle Aspirates (FNA) to interrogate PT responses in individuals originally primed with either wP or aP to assess differential germinal center (GC) Tfh cell and GC B cell responses in LNs of vaccine recipients, define whether differential antibody isotype responses of wP- vs aP-primed individuals is due to biases in GC Tfh helper functions to B cells and determine if circulating CD4+ T cells correlate with the vaccine immune response in the draining LN, the primary immune response site. In conclusion, we believe that results from this study will illuminate molecular reasons for lesser aP efficacy and illustrate human CD4+ T cell biological principles generally applicable to vaccinology and immunology.