Modeling distinct neonatal purine metabolism to inform vaccine development

描述（由申请人提供）：新生儿和婴儿有感染风险，对疫苗的反应不佳，部分原因是抗原呈递细胞和淋巴细胞的th1极化反应受损。对潜在机制的描述可以确定新生儿免疫的新方法。我们已经证明新生儿血浆中有较高浓度的腺苷（Ado），这是一种内源性嘌呤代谢产物，通过Ado受体起作用，增加胞质环Ado单磷酸（cAMP），从而损害th1极化免疫反应。新生儿血浆中含有高浓度的Ado生成碱性磷酸酶（AP）和5′外核苷酸酶CD73，而Ado脱氨酶（ADA）含量低于成人血浆，导致出生时Ado浓度较高。单核细胞（Mo）衍生的树突状细胞（MoDCs）对Ado抑制敏感，并表达细胞相关的Ado生成（CD73）和代谢（ADA）酶。为了更好地模拟内源性ado对新生儿免疫反应的调节，我们开发了一种自体三维新生儿组织构建体（NTC）和相应的成人组织构建体（ATC），由细胞外基质、自体血浆和覆盖的内皮组成，在其上可以研究冷冻保存的脐带血或外周血中单个核细胞或cd33选择的Mos外渗和自主MoDC的发育和激活。该项目的目标是通过追求以下具体目标来表征新生儿嘌呤代谢对佐剂（包括toll样受体激动剂和疫苗）免疫反应的影响：在目的1中，我们将评估AP、CD73和ADA对新生儿血浆中Ado积累的相对贡献。我们将采用薄层色谱，药物抑制剂，酶免疫消耗和高效液相色谱。嘌呤代谢酶的个体发育将使用新生儿和婴儿血浆样本进行表征。在Aim 2中，我们将确定可溶性和细胞相关的AP、CD73和ADA对自主生成的MoDCs激活的影响。我们将采用新的NTC，通过迁移、共刺激分子上调和Th1/Th2/Th17极化细胞因子的诱导，研究佐剂和疫苗（如卡介苗、乙肝疫苗和肺炎结合疫苗）诱导ADA、调节Ado水平和触发成熟MoDCs的分化和激活的能力。在Aim 3中，我们将在体外研究ag难治佐剂、TLR8激动剂和ADA对ag特异性新生儿疫苗反应的增强作用。我们将采用NTC衍生的modc和自体淋巴细胞共同培养，使用NTC进行疫苗抗原启动、增强和激发试验。这些研究将深入了解新生儿免疫，为研究免疫个体发生建立新的平台，评估难降解佐剂，并为新生儿疫苗的开发提供信息。

DESCRIPTION (provided by applicant): Newborns and infants are at risk of infection and respond sub-optimally to vaccines due in part to impaired Th1-polarizing responses of antigen-presenting cells and lymphocytes. Characterization of the underlying mechanisms may identify novel approaches to neonatal immunization. We have shown that neonatal blood plasma has higher concentrations of adenosine (Ado), an endogenous purine metabolite that acts via Ado receptors to increase cytosolic cyclic Ado mono-phosphate (cAMP) and thereby impairs Th1-polarizing immune responses. Neonatal plasma contains high concentrations of Ado-generating alkaline phosphatase (AP) and 5' ectonucleotidase CD73 while containing less Ado deaminase (ADA) than adult plasma, resulting in higher Ado concentrations at birth. Monocyte (Mo)-derived dendritic cells (MoDCs) are sensitive to Ado inhibition and express cell-associated Ado-generating (CD73) and metabolizing (ADA) enzymes. To better simulate endogenous Ado-based regulation of neonatal immune responses, we have developed an autologous three-dimensional neonatal tissue construct (NTC) and a corresponding adult tissue construct (ATC), comprised of extracellular matrix, autologous plasma, and an overlying endothelium upon which cryopreserved cord blood- or peripheral blood-mononuclear cells or CD33-selected Mos extravasate and autonomous MoDC development and activation can be studied. The project objective is to characterize the impact of neonatal purine metabolism on immune responses to adjuvants, including Toll-like receptor (TLR) agonists, and vaccines, by pursuing the following Specific Aims: In Aim 1, we will assess the relative contribution of AP, CD73 and ADA to Ado accumulation in human neonatal vs. adult blood plasma. We will employ thin layer chromatography, pharmacologic inhibitors, enzyme immunodepletion, and high performance liquid chromatography. The ontogeny of purine-metabolizing enzymes will be characterized using neonatal and infant plasma samples. In Aim 2, we will determine effects of soluble and cell-associated AP, CD73 and ADA on activation of autonomously generated MoDCs. We will employ the novel NTC, interrogating the ability of adjuvants and vaccines, such as Bacillus Calmette-Guerin (BCG), hepatitis B vaccine, and pneumoccal conjugate vaccine to induce ADA, modulate Ado levels, and trigger differentiation and activation of mature MoDCs as measured by migration, up-regulation of co-stimulatory molecules, and induction of Th1/Th2/Th17- polarizing cytokines. In Aim 3, we will characterize enhancing effects of Ado-refractory adjuvants, TLR8 agonists and ADA, on Ag-specific neonatal vaccine responses in vitro. We will employ co-culture of NTC-derived MoDCs and autologous lymphocytes in vaccine antigen prime, boost, and challenge assays using the NTC. These studies will provide insight into neonatal immunity, establish novel platforms for studying immune ontogeny, assess Ado-refractory adjuvants, and inform neonatal vaccine development.