Influenza pathogenesis in pregnancy

项目摘要 孕妇非常容易感染甲型流感病毒(IAV)，产妇死亡的风险增加， 早产和死产。通用流感疫苗(UFV)被认为是可能的，如果保守的区域 流感病毒是有针对性的，并产生适当的免疫反应。然而，相关的动物模型是 缺乏测试这种疫苗的能力，特别是对怀孕妇女。这项建议的重点是调查 妊娠非人灵长类(NHP)模型中母体和胎盘对IAV的免疫反应 病毒宿主因素导致母体疾病加重。我们的中心假设是异常的Th17反应 在急性IAV感染期间，会导致广泛功能失调的先天性和获得性免疫反应 阻止病毒清除，增加产妇死亡和死产的风险。Th17细胞产生高水平的 炎性细胞因子IL-17和IL-2218，19与早期Th17极化被认为是IAV的关键 在小鼠模型中，IL-23异常和/或延迟激活Th17通路被认为会损害病毒 清除和促进肺损伤。20，21我们在妊娠NHP急性IAV H1N1感染模型中的初步数据 显示所有动物在接种IAV后第5天出现肺炎。在怀孕的NHP中，流感疾病评分 明显肺外脏器损伤(心肌炎、白质损伤)的非妊娠动物。 妊娠NHP患者外周血与PBMC偶联的早期Th17CD4T细胞反应几乎缺失 免疫病理高峰期肺内Th17细胞较非妊娠动物明显增多。 肺和支气管肺泡灌洗液(BAL)中的炎性细胞因子和趋化因子也较高。 怀孕的动物与未怀孕的动物。在目标1中，未怀孕和怀孕的辫子猕猴将受到挑战 使用IAV H1N1 A/CA/04/09或H3N2A/Texas/71/2017(N=8，每组)，并接受血液和BAL 取样至第5天尸检(免疫病理学高峰期)。在目标1A和1B中，我们将确定怀孕- 通过评估Th17、CD4T细胞和广谱细胞的频率与IAV病的特异性免疫相关性 先天/获得性免疫反应(即免疫细胞亚群、细胞因子/趋化因子、I/III型干扰素) 血液、BAL和肺。在AIM 1C中，我们将评估与不良妊娠有关的胎盘的抗病毒反应 结果(如细胞因子/趋化因子、NLRP3炎症小体激活、CD8T细胞)。在目标2中，我们将使用批量 和单细胞RNA测序以确定PBMC、BAL、肺和胎盘中转录组的变化 关注Th17转录网络和抗病毒的先天免疫途径。总而言之，初步数据 表明怀孕动物体内存在异常的Th17反应，这对促进病毒清除和 预防肺损伤。这些研究将首次全面分析先天/获得性免疫反应。 在一次急性IAV感染期间，以阐明孕妇严重肺部疾病的发病机制。结果来自 这些研究对于IAV大流行的准备工作至关重要，以便能够测试新的UFV的有效性和安全性。

Project Summary Pregnant women are highly vulnerable to influenza A virus (IAV) and are at increased risk for maternal death, preterm birth and stillbirth. Universal influenza vaccines (UFV) are thought to be possible if conserved regions of influenza virus are targeted and appropriate immune responses generated. However, relevant animal models are lacking in which to test such a vaccine, particularly for pregnant women. This proposal is focused on investigating maternal and placental immune responses to IAV in a pregnant nonhuman primate (NHP) model to understand the viral-host factors driving enhanced maternal disease. Our central hypothesis is that an aberrant Th17 response during an acute IAV infection leads to a broadly dysfunctional innate and adaptive immune response that prevents viral clearance and enhances risk for maternal death and stillbirth. Th17 cells produce high levels of the inflammatory cytokines IL-17 and IL-2218,19 with early Th17 polarization considered to be critical for IAV resolution; aberrant and/or late activation of the Th17 pathway by IL-23 in murine models is thought to impair viral clearance and promote lung injury.20,21 Our preliminary data in a pregnant NHP model of an acute IAV H1N1 infection demonstrates pneumonia in all animals by Day 5 post-IAV inoculation. In pregnant NHP, influenza disease scores were higher than non-pregnant animals with notable extra-pulmonary organ injury (myocarditis, white matter injury). Pregnant NHP demonstrated a nearly absent early Th17 CD4+ T cell response in whole blood and PBMC coupled with a marked increase in Th17 cells in the lung at peak immunopathology compared to non-pregnant animals. Inflammatory cytokines and chemokines in the lungs and bronchoalveolar lavage fluid (BAL) were also greater in pregnant versus non-pregnant animals. In Aim 1, non-pregnant and pregnant pigtail macaques will be challenged with either IAV H1N1 A/CA/04/09 or H3N2 A/Texas/71/2017 (N=8, each group) and undergo blood and BAL sampling until necropsy at Day 5 (peak immunopathology). In Aims 1A and 1B, we will determine pregnancy- specific immune correlates of IAV disease by evaluating the frequency of Th17 CD4+ T cells and a broad spectrum of innate/adaptive immune responses (i.e. immune cell subsets, cytokines/chemokines, Type I/III interferons) in the blood, BAL and lung. In Aim 1C, we will evaluate antiviral responses in the placenta linked to adverse pregnancy outcomes (e.g. cytokines/chemokines, NLRP3 inflammasome activation, CD8+ T cells). In Aim 2, we will use bulk and single cell RNA-sequencing to define changes in the transcriptome within PBMC, BAL, lung and placenta with a focus on Th17 transcriptional networks and antiviral innate immune pathways. In summary, the preliminary data indicates an aberrant Th17 response in pregnant animals, which is critical to promoting viral clearance and preventing lung injury. These studies will be the first to comprehensively analyze innate/adaptive immune responses during an acute IAV infection to elucidate the pathogenesis of severe lung disease in pregnant women. Results from these studies are critical for IAV pandemic preparedness to enable testing of efficacy and safety of new UFV.