Modeling distinct neonatal purine metabolism to inform vaccine development

模拟不同的新生儿嘌呤代谢为疫苗开发提供信息

• 批准号: 8532818
• 负责人: OFER LEVY
• 金额: $ 42.63万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-16 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8532818
• 关键词: 5' -Nucleotidase; Adenosine; Adenylate Cyclase; Adjuvant; Adult; Agonist; Alkaline Phosphatase; Anti-Inflammatory Agents; Anti-inflammatory; Antigen-Presenting Cells; Antigens; Autologous; Biological Assay; Biological Products; Birth; Blood; Calmette-Guerin Bacillus; Cells; Coculture Techniques; Conjugate Vaccines; Cyclic AMP; Data; Deaminase; Dendritic Cells; Development; Dose; Drug Formulations; Endothelium; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Exposure to; Extracellular Matrix; Frequencies; Goals; Growth; Healthcare; Heating; Hepatitis B Vaccines; High Pressure Liquid Chromatography; Human; Immune; Immune response; Immunity; Immunization; In Vitro; Infant; Infection; Inflammatory Response; Inosine; Left; Leukocytes; Life; Lymphocyte; Measures; Medical; Modeling; Mono-S; Neonatal; Newborn Infant; Peripheral Blood Mononuclear Cell; Physiological; Plasma; Predisposition; Property; Public Health; Publishing; Purinergic P1 Receptors; Purines; Refractory; Regulation; Relative (related person); Research; Risk; Sampling; Second Messenger Systems; Serum; Severities; Simulate; TLR8 gene; Testing; Thin Layer Chromatography; Tissues; Toll-like receptors; Umbilical Cord Blood; Up-Regulation; Vaccination; Vaccine Adjuvant; Vaccine Antigen; Vaccines; adaptive immunity; adenosine deaminase; autologous lymphocytes; base; cytokine; infancy; inorganic phosphate; insight; lymphocyte proliferation; migration; monocyte; neonatal human; novel; novel strategies; purine; purine metabolism; response; second messenger; vaccine development

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.

描述（由申请人提供）：新生儿和婴儿面临感染风险，并且对疫苗的反应欠佳，部分原因是抗原呈递细胞和淋巴细胞的 Th1 极化反应受损。潜在机制的表征可能会确定新生儿免疫的新方法。我们已经证明，新生儿血浆中含有较高浓度的腺苷 (Ado)，这是一种内源性嘌呤代谢物，通过 Ado 受体发挥作用，增加胞质环 Ado 单磷酸 (cAMP)，从而损害 Th1 极化免疫反应。新生儿血浆含有高浓度的 Ado 生成碱性磷酸酶 (AP) 和 5' 核酸外切酶 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) 衍生的树突细胞 (MoDC) 对 Ado 抑制敏感，并表达细胞相关的 Ado 生成 (CD73) 和代谢 (ADA) 酶。为了更好地模拟新生儿免疫反应的内源性 Ado 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、自体血浆和覆盖在其上的内皮细胞组成。可以研究冷冻保存的脐带血或外周血单核细胞或 CD33 选择的 Mos 渗出液以及自主 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 (TLR) 激动剂和疫苗）免疫反应的影响：在目标 1 中，我们将评估 AP 的相对贡献、CD73 和 ADA 到 Ado 在人类新生儿与成人血浆中的积累。我们将采用薄层色谱法、药物抑制剂、酶免疫耗竭和高效液相色谱法。将使用新生儿和婴儿血浆样本来表征嘌呤代谢酶的个体发育。在目标 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 MoDC 激活的影响。我们将采用新型 NTC，探究佐剂和疫苗（例如卡介苗 (BCG)、乙型肝炎疫苗和肺炎球菌结合疫苗）诱导 ADA、调节 Ado 水平并触发成熟 MoDC 分化和激活的能力。通过迁移、共刺激分子的上调以及 Th1/Th2/Th17 极化细胞因子的诱导来测量。在目标 3 中，我们将描述 Ado 难治性佐剂、TLR8 激动剂和 ADA 对体外 Ag 特异性新生儿疫苗反应的增强作用。我们将在使用 NTC 的疫苗抗原初免、加强和攻击检测中采用 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 Ado 难治性佐剂，并为新生儿疫苗的开发提供信息。