Defining molecular mechanisms of combination adjuvants: a systems immunology, transcriptomics and imaging approach

项目摘要/摘要 在这个提案中，我们将定义已知佐剂的不同组合的分子和细胞机制 组分Mpla(TL4激动剂)、CpG(TLR9激动剂)、cGAS-STING和NOD1/2通路激动剂， 和角鲨烯水乳剂(AddaVAX™)。我们的总体目标是提供对每个 结合使用高通量的体外和体内试验，随后对两个 联合佐剂活体细胞成像和单细胞mRNA测序检测术后引流淋巴结 接种疫苗。 最初，基于96孔的天然免疫系统和获得性免疫系统激活的体外分析将被用来分析 不同的佐剂成分，既有单独的，也有不同的组合和浓度。这些 检测将包括：1)使用原代树突状细胞激活TLR、NOD和STING信号通路 报告转基因小鼠(DC)、T细胞和B细胞；2)体外激活幼稚B细胞以监测其 分化为浆细胞和班级转换。我们预计一些佐剂组合将具有 当单独使用时，不同于效果总和的协同效应。接下来，基于 体外联合佐剂的子集及其单独的成分将作为模型的佐剂进行评估 体内疫苗抗原(流感血凝素H1、丝状病毒(EBOV)糖蛋白和SARS-CoV-2尖峰) 老鼠。免疫原性指标将包括：1)抗体的动态和持久性、同型、亲和力和广度 利用蛋白质微阵列的交叉反应；2)抗原特异性B细胞的流式细胞术以评估分化 和交叉反应；3)T细胞召回试验以确定Th1/Th2/Th17细胞因子谱；4)血清中和 流感、SARS-CoV-2和VSV-EBOV糖蛋白假型。这将提供一个全面的 与每种组合佐剂相关的细胞和分子图谱。 将选择两种组合佐剂(及其单独的成分进行比较)进行深入分析 使用：1)转基因小鼠，允许活的CD4T和B细胞以及DC中的钙流动使用2- 光子显微镜。结合全组织成像技术，我们将监测佐剂驱动的T细胞 和DC在活体引流淋巴结中的动员、运动和相互作用；2)使用单细胞RNAseq 技术(10倍基因组公司)引流淋巴结中的细胞，我们将定义细胞组成和表型， 细胞相互作用和空间组织。我们将在第一年对这一组合进行深入分析 佐剂CpG/Mpla AddaVAX(角鲨烯水乳剂中的TLR9和TLR4激动剂) 已经证明这是一种强有力的联合佐剂。将选择第二种组合佐剂用于深度治疗 基于在这里描述的体外和体内检测中产生的数据的分析。把这些放在一起 互补的深度方法将提供前所未有的分子和细胞细节水平 高效复合佐剂。总的来说，我们预计这些数据将有助于指导未来的设计 需要免疫反应的疫苗可以根据特定的病原体进行调整。

PROJECT SUMMARY/ABSTRACT In this proposal we will define molecular and cellular mechanisms of different combinations of known adjuvant components MPLA (a TL4 agonist), CpG (a TLR9 agonist), agonists of cGAS-STING and NOD1/2 pathways, and a squalene-in-water emulsion (AddaVAX™). Our overall Aim is to provide a detailed analysis of every combination of these using high throughput in vitro and in vivo assays, followed an in-depth analysis of two combination adjuvants using live cell imaging and single-cell mRNA sequencing of draining lymph nodes after vaccination. Initially, 96-well based in vitro assays of innate and adaptive immune system activation will be used to profile different adjuvants components, both individually, and in different combinations and concentrations. These assays will comprise: 1) activation of TLR, NOD and STING signaling pathways using primary dendritic cells (DCs), T and B cells from reporter transgenic mice; 2) in vitro activation of naïve B cells to monitor their differentiation into plasma cells and class switching. We anticipate some of the adjuvant combinations will have synergistic effects that differ from the sum of the effects when used individually. Next, based on performance in vitro a subset of combination adjuvants and their individual components will be evaluated as adjuvants for model vaccine antigens (influenza hemagglutinin H1, filovirus (EBOV) glycoprotein and SARS-CoV-2 spike) in vivo in mice. Immunogenicity metrics will comprise: 1) antibody dynamics and durability, isotype, avidity and breadth of cross-reactivity using protein microarrays; 2) flow cytometry of antigen-specific B cells to assess differentiation and cross-reactivity; 3) T cell recall assays to define Th1/Th2/Th17 cytokine profiles; 4) neutralization by sera of live influenza, SARS-CoV-2 and VSV-pseudotyped with EBOV glycoprotein. This will provide a comprehensive cellular and molecular profile associated with each combination adjuvant. Two combination adjuvants (and their individual components for comparison) will be selected for a deep analysis using: 1) transgenic mice that allow Ca2+ fluxes in live CD4 T and B cells and DCs to be visualized using 2- photon microscopy. Combined with techniques of whole tissue imaging, we will monitor adjuvant-driven T cell and DC mobilization, motility and interactions in live draining lymph nodes; 2) using single-cell RNAseq technology (10x Genomics Inc) of cells in draining lymph nodes, we will define cell composition and phenotype, cellular interactions and spatial organization. We will perform a deep analysis in Year 1 on the combination adjuvant CpG/MPLA + AddaVAX (TLR9 and TLR4 agonists in a squalene-in-water emulsion) since we have already shown this is a powerful combination adjuvant. A second combination adjuvant will be selected for deep analysis based on data generated in the in vitro and in vivo assays described herein. Together these complementary deep approaches will provide an unprecedented level of molecular and cellular detail of two highly effective combination adjuvants. Overall, we anticipate these data will help guide the future design of vaccines where the immune response required can be tuned according to the particular pathogen in question.