Influenza pathogenesis in pregnancy

项目摘要 孕妇非常容易感染甲型流感病毒（IAV），孕产妇死亡的风险增加， 早产和死胎。通用流感疫苗（UFV）被认为是可能的，如果保守的区域， 靶向流感病毒并产生适当免疫应答。然而，相关的动物模型是 缺乏测试这种疫苗的方法，特别是对孕妇。这项建议的重点是调查 妊娠非人灵长类动物（NHP）模型中母体和胎盘对IAV的免疫应答，以了解 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Th 17应答 在急性IAV感染期间，导致先天性和适应性免疫应答功能广泛失调， 阻止病毒清除，增加产妇死亡和死产的风险。Th 17细胞产生高水平的 具有早期Th 17极化的炎性细胞因子IL-17和IL-2218，19被认为是IAV的关键 在小鼠模型中，IL-23对Th 17通路的异常和/或晚期激活被认为损害了病毒的免疫应答。 清除并促进肺损伤。20，21我们在急性IAV H1N1感染的妊娠NHP模型中的初步数据 在IAV接种后第5天在所有动物中证实肺炎。在妊娠NHP中，流感疾病评分 高于非妊娠动物，并伴有显著的肺外器官损伤（心肌炎、白色物质损伤）。 妊娠NHP在全血和PBMC偶联中几乎不存在早期Th 17 CD 4 + T细胞应答， 与未怀孕的动物相比，在免疫病理学高峰时肺中的Th 17细胞显著增加。 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高于对照组。 妊娠与非妊娠动物。在目标1中，将对非妊娠和妊娠猪尾猕猴进行攻毒 感染IAV H1N1 A/CA/04/09或H3 N2 A/Texas/71/2017（每组N=8），并接受血液和BAL 采样直至第5天尸检（免疫病理学峰值）。在目标1A和1B中，我们将确定怀孕- 通过评估Th 17 CD 4 + T细胞的频率和广谱免疫相关性， 先天性/适应性免疫应答（即免疫细胞亚群，细胞因子/趋化因子，I/III型干扰素）的 血液BAL和肺在目标1C中，我们将评估与不良妊娠相关的胎盘抗病毒反应 结果（例如细胞因子/趋化因子、NLRP 3炎性小体活化、CD 8 + T细胞）。在目标2中，我们将使用bulk 和单细胞RNA测序，以确定PBMC、BAL、肺和胎盘内转录组的变化， 重点是Th 17转录网络和抗病毒先天免疫途径。总之，初步数据 表明妊娠动物中异常的Th 17应答，这对促进病毒清除至关重要， 防止肺损伤。这些研究将首次全面分析先天/适应性免疫反应 在急性IAV感染期间，以阐明孕妇严重肺部疾病的发病机制。结果 这些研究对于IAV大流行的准备是至关重要的，从而能够测试新的UFV的功效和安全性。

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.