Fetal Zika virus infection: role of the human placenta

项目摘要/摘要 寨卡疫情始于巴西，现已蔓延至中美洲和美国领土，由 据疾控中心报告，亚洲寨卡病毒(ZIKV)株与严重的出生缺陷有关。 小头畸形、流产和死产。ZIKV RNA在胎盘、胎儿和母体中的检测 循环数月，与长期感染相关的钙化，以及宫内生长受限 表明病毒诱导的病理损害了胎盘功能。解决经胎盘ZIKV的问题 在传播方面，我们提议在加州大学勒诺尔·佩雷拉博士和加州大学伊娃·哈里斯博士之间开展一个合作项目 伯克利)，在人类先天性感染方面具有互补专业知识的经验丰富的调查人员 胎盘和黄病毒研究。我们的初步研究表明，原型和最近的iso- 尼加拉瓜ZIKV新毒株感染胎盘组织中的细胞和人胎盘原代细胞 表达Ax1、Tyro3和TIM1酪氨酸激酶受体，介导ZIKV和密切相关的 皮肤感染登革病毒(DENV)。感染的胎盘细胞，包括胎儿羊膜上皮细胞，胎盘 成纤维细胞和滋养层祖细胞，发展为细胞病理学，表达ZIKV包膜和非 NS3蛋白的结构和释放的病毒滴度取决于表达的受体和胎龄。 AXL在蜕膜(子宫蜕膜细胞和侵袭性细胞滋养细胞)中检测到，表明感染途径。 绒毛(胎盘成纤维细胞、霍夫鲍尔细胞和血管)和胎膜(羊膜上皮) 细胞和滋养层祖细胞)。受体的差异表达提示ZIKV是如何感染 从蜕膜扩散到胎盘、胎儿和羊膜。此外，在流行地区，交叉- 预先存在的反应性抗体可能在ZIKV的保护或致病中发挥关键作用 妊娠期间的胎盘组织。我们的主要假设是胎盘原代细胞和外植体 蜕膜、绒毛和羊膜可作为人体胎盘的模型。 确定ZIKV由游离病毒粒子感染的分子机制；抗体在中和、反式病毒感染中的作用 胎盘中ZIKV感染的细胞增多和增强；以及抑制剂和Thera-2的翻译影响。 常规中和单抗(MAb)。我们的具体目标是：目标1.鉴定子宫细胞- 原代细胞和胎盘中易受尼加拉瓜寨卡病毒株感染的胎盘界面 外植体，同源受体的特征，并识别感染的小分子抑制物。目标2.研究 ZIKV特异性抗体和DENV交叉反应抗体在感染中的中和和潜在增强作用 和原代胎盘细胞和外植体的细胞转化，并确定中和的治疗潜力 带有修饰的Fc的单抗，排除了抗体依赖的增强。拟议的研究将揭示 寨卡病毒感染的分子机制，病毒传播到胎儿的途径，以及治疗的可能性 经过改造的抗体和小分子抑制剂，可以阻止感染和预防先天性疾病。

PROJECT SUMMARY/ABSTRACT The Zika epidemic that began in Brazil and has spread to Central America and US territories, caused by the Asian Zika virus (ZIKV) strain, has been reported by the CDC as definitively linked to severe birth defects – microcephaly, miscarriage and stillbirth. Detection of ZIKV RNA in the placenta and fetus and in maternal circulation for months, calcifications associated with long-standing infection, and intrauterine growth restriction indicate that virus-induced pathology impairs placental functions. To address the issue of transplacental ZIKV transmission, we propose a collaborative project between Dr. Lenore Pereira (UCSF) and Dr. Eva Harris (UC Berkeley), experienced investigators with complementary expertise in congenital infection of the human placenta and Flavivirus research, respectively. Our preliminary studies reveal that prototype and recently iso- lated Nicaraguan ZIKV strains infect cells in placental explants and primary cells isolated from human placenta that express AXL, Tyro3 and TIM1 tyrosine kinase receptors, which mediate ZIKV and the closely related dengue virus (DENV) infection in skin. Infected placental cells, including fetal amniotic epithelial cells, placental fibroblasts and trophoblast progenitor cells, developed cytopathology and expressed ZIKV envelope and non- structural NS3 proteins, and virus titers released depended on the receptors expressed and gestational age. Indicative of infection route, AXL was detected in decidua (uterine decidual cells and invasive cytotrophoblasts), chorionic villi (placental fibroblasts, Hofbauer cells and blood vessels) and fetal membranes (amniotic epithelial cells and trophoblast progenitor cells). Differential expression of receptors suggests how ZIKV could infect the decidua and spread to the placenta, fetus and amnion-chorion membranes. Further, in endemic regions, cross- reactive pre-existing antibodies to DENV could play a critical role in protection or pathogenesis of ZIKV in placenta tissues during pregnancy. Our overarching hypothesis is that placental primary cells and explants of decidua, chorionic villi and amnion-chorion membranes can be used as a model of the human placenta to define molecular mechanisms of ZIKV infection by free virions; the role of antibodies in neutralization, trans- cytosis, and enhancement of ZIKV infection in the placenta; and the translational impact of inhibitors and thera- peutic neutralizing monoclonal antibodies (MAbs). Our Specific Aims are: Aim 1. Identify cells at the uterine- placental interface that are susceptible to infection by Nicaraguan ZIKV strains in primary cells and placental explants, characterize cognate receptors, and identify small-molecule inhibitors of infection. Aim 2. Study the neutralizing and potentially enhancing role of ZIKV-specific and DENV-cross-reactive antibodies on infection and transcytosis in primary placental cells and explants and determine the therapeutic potential of neutralizing MAbs with modified Fc that preclude antibody-dependent enhancement. The proposed studies will reveal molecular mechanisms of ZIKV infection, routes of virus transmission to the fetus, and therapeutic potential for engineered antibodies and small molecule inhibitors that block infection and prevent congenital disease.