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的风险，对疫苗的反应不佳，部分原因是抗原提呈细胞和淋巴细胞的Th1极化反应受损。对潜在机制的描述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受体作用于胞浆中的环磷酸腺苷(CAMP)，从而损害Th1极化的免疫反应。新生儿血浆中含有较高浓度的腺苷生成碱性磷酸酶(AP)和5‘-核糖核酸酶CD73，而腺苷脱氨酶(ADA)含量低于成人血浆，导致出生时的腺苷浓度较高。单核细胞来源的树突状细胞(MoDC)对腺苷抑制反应敏感，表达细胞相关的腺苷生成(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，我们开发了一个自体三维新生儿组织结构(NTC)和一个相应的成人组织结构(ATC)，该结构由细胞外基质、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血或外周血单核细胞或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)免疫反应的影响：在目标1中，我们将评估AP、CD73和ADA对新生儿和成人血浆中ADO积累的相对贡献。我们将使用薄层层析、药物抑制剂、酶免疫耗竭和高效液相色谱。将使用新生儿和婴儿的血浆样本来描述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AP、CD73和ADA对自主产生的MoDC激活的影响。我们将使用新的NTC，询问佐剂和疫苗，如卡介苗(BCG)，乙肝疫苗和肺炎结合疫苗诱导ADA，调节ADO水平，并通过迁移、上调共刺激分子和诱导Th1/Th2/Th17极化细胞因子来触发成熟MoDC的分化和激活的能力。在目标3中，我们将表征ADO-难治性佐剂、TLR8激动剂和ADA在体外对抗原特异性新生儿疫苗应答的增强作用。我们将使用NTC来源的MODC和自体淋巴细胞在疫苗抗原初始、增强和挑战试验中使用NTC进行共培养。这些研究将提供对新生儿免疫的洞察，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。 公共卫生相关性：每年有200多万新生儿和幼儿死于感染，部分原因是他们的免疫反应相对较弱。我们的项目利用血液成分来模拟新生儿和婴儿对疫苗的免疫反应。通过研究生命早期影响免疫反应的机制和因素，我们希望找到更有效的方法来为新生儿接种疫苗，从而保护他们免受感染，提高公共卫生水平。

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. PUBLIC HEALTH RELEVANCE: Over 2,000,000 newborns and young infants die due to infection every year, in part due to their relatively weak immune responses. Our project makes use of blood components to model immune responses of newborns and infants to vaccines. By studying mechanisms and factors that impair immune responses early in life, we hope to identify more effective ways to immunize newborns, thereby protecting them from infection and enhancing public health.