Modeling distinct neonatal purine metabolism to inform vaccine development

描述（由申请方提供）：新生儿和婴儿有感染风险，部分由于抗原呈递细胞和淋巴细胞的Th 1极化反应受损，对疫苗的反应不佳。潜在机制的表征可能会发现新生儿免疫的新方法。我们已经表明，新生儿血浆中有较高浓度的腺苷（Ado），内源性嘌呤代谢产物，通过Ado受体的作用，以增加细胞溶质环腺苷单磷酸（cAMP），从而削弱Th 1极化免疫反应。新生儿血浆中含有高浓度的腺苷酸生成碱性磷酸酶（AP）和5'外核苷酸酶CD 73，而含有比成人血浆更少的腺苷脱氨酶（ADA），导致出生时较高的腺苷浓度。单核细胞（Mo）衍生的树突状细胞（MoDC）对Ado抑制敏感，并表达细胞相关的CD 73生成酶和代谢酶（ADA）。为了更好地模拟新生儿免疫应答的基于内源性TNF的调节，我们开发了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），其由细胞外基质、自体血浆、和覆盖内皮，在其上冷冻保存的脐带血或外周血单核细胞或CD 33-可以研究选择的MoS外渗物和自主的MoDC发育和活化。该项目的目标是通过追求以下特定目标来表征新生儿嘌呤代谢对佐剂（包括Toll样受体（TLR）激动剂）和疫苗免疫应答的影响：在目标1中，我们将评估AP、CD 73和ADA对人类新生儿与成人血浆中Ado蓄积的相对贡献。我们将采用薄层层析、药理学抑制剂、酶免疫耗竭和高效液相层析。将使用新生儿和婴儿血浆样本表征嘌呤代谢酶的个体发生。在目标2中，我们将确定可溶性和细胞相关的AP、CD 73和ADA对自主产生的MoDC的活化的影响。我们将采用新的NTC，询问佐剂和疫苗的能力，如卡介苗（BCG），乙肝B疫苗，和大肠杆菌缀合物疫苗诱导ADA，调节Ado水平，并触发分化和激活成熟的MoDC的迁移，上调共刺激分子，诱导Th 1/Th 2/Th 17极化细胞因子。在目标3中，我们将表征免疫耐受性佐剂、TLR 8激动剂和ADA对体外Ag特异性新生儿疫苗应答的增强作用。我们将在使用NTC的疫苗抗原初免、加强和激发测定中采用NTC衍生的MoDC和自体淋巴细胞的共培养。这些研究将提供对新生儿免疫的深入了解，建立研究免疫个体发育的新平台，评估免疫耐受性佐剂，并为新生儿疫苗开发提供信息。

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.