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

项目概要/摘要 在这个建议中，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（TL 4激动剂），CpG（TLR 9激动剂），cGAS-STING和NOD 1/2途径的激动剂， 和水包角鲨烯乳液（AddaVAX™）。我们的总体目标是提供一个详细的分析， 这些使用高通量体外和体内测定的组合，随后对两个 使用活细胞成像和引流淋巴结的单细胞mRNA测序的组合佐剂 预防针 最初，将使用基于96孔的先天性和适应性免疫系统激活的体外测定来分析 不同的佐剂组分，无论是单独的，还是不同的组合和浓度。这些 测定将包括：1）使用原代树突细胞激活TLR、NOD和STING信号传导途径 (DCs)、T和B细胞; 2）体外活化幼稚B细胞以监测它们的免疫应答; 分化成浆细胞和类别转换。我们预计一些佐剂组合将有 协同效应不同于单独使用时的效应总和。接下来，根据在 体外将评估一组组合佐剂及其单独组分作为模型的佐剂 疫苗抗原（流感血凝素H1、丝状病毒（EBOV）糖蛋白和SARS-CoV-2刺突）， 小鼠免疫原性指标将包括：1）抗体动力学和持久性、同种型、亲合力和免疫原性的广度。 使用蛋白质微阵列的交叉反应性; 2）抗原特异性B细胞的流式细胞术以评估分化 和交叉反应性; 3）确定Th 1/Th 2/Th 17细胞因子谱的T细胞回忆测定; 4）通过免疫球蛋白的血清中和， 活流感病毒、SARS-CoV-2和用EBOV糖蛋白假型化的VSV。这将提供全面的 与每种组合佐剂相关的细胞和分子谱。 将选择两种组合佐剂（及其用于比较的单个组分）进行深入分析 使用：1）转基因小鼠，其允许活的CD 4 T和B细胞和DC中的Ca 2+通量使用2- 光子显微镜结合全组织成像技术，我们将监测趋化性T细胞 和DC动员，运动性和相互作用在活引流淋巴结; 2）使用单细胞RNAseq 技术（10 x Genomics Inc）的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。我们将在第一年对组合进行深入分析 佐剂CpG/MPLA + AddaVAX（TLR 9和TLR 4激动剂在水包角鲨烯乳剂中），因为我们已经 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免疫。 基于本文所述的体外和体内测定中产生的数据进行分析。综合这些 互补的深度方法将提供前所未有的分子和细胞细节， 高效组合佐剂。总的来说，我们预计这些数据将有助于指导未来的设计， 疫苗，其中所需的免疫反应可以根据所讨论的特定病原体进行调整。

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.