Combination Adjuvants to Activate Human Dendritic Cell Subsets and B Cells

 说明（申请人提供）：接种疫苗是预防传染病最有效的方法。许多目前的疫苗是由纯化的或重组的病原体组分组成的“灭活”或“亚单位”疫苗，通常向其中加入佐剂以增加抗体应答的幅度。然而，用目前FDA批准的佐剂配制的亚单位疫苗在某些人群中不能充分增强免疫力，特别是免疫功能低下和老年受试者。近年来，已经发现了许多佐剂，并且作为单一药剂显示出增强的免疫原性;然而，关于它们的组合的活性、它们的安全性、它们的功效和它们的机制知之甚少 的行动。对疫苗接种和佐剂的应答涉及树突状细胞（DC），其捕获并呈递疫苗抗原，从而促进滤泡辅助T细胞（Tfh）和B细胞的分化以及随后的体液免疫。因此，我们建议检查暴露于组合佐剂的人DC亚群的分子机制和功能输出离体和体内。考虑到小鼠和人之间先天免疫受体分布和功能的实质性差异，对人DC的关注是必不可少的。我们的目标是选择一个组合佐剂使用功能测定，然后深入调查的分子途径占增强免疫原性。我们与工业界的合作将使选定的组合佐剂过渡到人类疫苗接种的进一步研究。我们的具体目标就是为了实现这一目标。因此，首先，我们将通过评估佐剂活化的人DC亚群使初始CD 4 +T细胞分化为分泌IL-21的Tfh细胞并诱导B细胞产生IgG和伊加抗体的能力来筛选佐剂组合（目的1）。将使用经验证的、敏感的和高通量的转录组学表观基因组学、蛋白质组学和代谢组学方法，并通过体外功能性敲低，在DC中进一步研究有前景的组合，以确定佐剂功效的潜在分子途径（目的2）。然后，我们将使用携带功能重建的人免疫系统的人源化小鼠，通过体内功能敲除研究验证所鉴定的分子途径，这也将能够检查可能的副作用（目的3）。拟议的研究计划将利用尖端的表观遗传（ATAC-seq），转录，基因编辑（CRISPR/Cas9）和代谢组学技术;创新的人源化小鼠模型;杰克逊实验室强大的计算和生物信息学基础设施;以及充满活力的研究团队的互补专业知识。我们的交付物是一种组合佐剂，用于增强体液免疫和对其功效至关重要的分子途径。

 DESCRIPTION (provided by applicant): Vaccination is the most effective method for preventing infectious diseases. Many current vaccines are "inactivated" or "subunit" vaccines composed of purified or recombinant pathogen components to which an adjuvant is often added to increase the magnitude of antibody responses. However, subunit vaccines formulated with current FDA-approved adjuvants do not sufficiently boost immunity in some populations, particularly immunocompromised and elderly subjects. Numerous adjuvants have been discovered in recent years and show enhanced immunogenicity as single agents; however, little is known about the activity of their combination, their safety, their efficacy and their mechanisms of action. Responses to vaccination and adjuvants involve dendritic cells (DCs), which capture and present vaccine antigens thereby facilitating the differentiation of follicular helper T cells (Tfh) and B cells and subsequent humoral immunity. Therefore, we propose to examine the molecular mechanisms and functional outputs of human DC subsets exposed to combination adjuvants ex vivo and in vivo. The focus on human DCs is essential given the substantial differences in innate immune receptor distribution and function between the mouse and the human. Our goal is to select a combination adjuvant using functional assays, followed by in-depth investigation of molecular pathways accounting for enhanced immunogenicity. Our collaboration with industry will enable the transition of the selected combination adjuvant to further studies of human vaccination. Our Specific Aims are built towards this goal. Thus, first we will screen adjuvant combinations by assessing the capacity of adjuvant-activated human DC subsets to skew the differentiation of naïve CD4+T cells into Tfh cells that secrete IL-21 and induce B cells to produce IgG and IgA antibodies (Aim 1). Promising combinations will be further studied in DCs using validated, sensitive and high-throughput transcriptomic epigenomic, proteomic and metabolomic methods, and by functional knockdown in vitro, to determine the underlying molecular pathways of adjuvant efficacy (Aim 2). We will then validate the identified molecular pathways through functional knockdown studies in vivo using humanized mice carrying a functionally reconstituted human immune system, which will also enable the examination of possible side effects (Aim 3). The proposed research program will leverage cutting-edge epigenetic (ATAC-seq), transcriptional, gene editing (CRISPR/Cas9) and metabolomic technologies; innovative humanized mouse models; a powerful computational and bioinformatics infrastructure at The Jackson Laboratory; and the complementary expertise of a dynamic team of investigators. Our deliverable is a combination adjuvant for enhanced humoral immunity and molecular pathways that are essential for its efficacy.