Influenza pathogenesis in pregnancy

项目概要 孕妇极易感染甲型流感病毒 (IAV)，孕产妇死亡的风险也会增加， 早产和死产。如果存在保守区域，通用流感疫苗（UFV）被认为是可能的。 流感病毒被靶向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方法，特别是针对孕妇。该提案的重点是调查 在怀孕的非人灵长类动物 (NHP) 模型中对 IAV 的母体和胎盘免疫反应，以了解 病毒宿主因素导致孕产妇疾病加剧。我们的中心假设是异常的 Th17 反应 在急性 IAV 感染期间，会导致先天性和适应性免疫反应广泛功能失调， 阻止病毒清除并增加孕产妇死亡和死产的风险。 Th17 细胞产生高水平 炎症细胞因子 IL-17 和 IL-2218,19 以及早期 Th17 极化被认为对 IAV 至关重要 决议；在小鼠模型中，IL-23 对 Th17 通路的异常和/或晚期激活被认为会损害病毒 清除并促进肺损伤。20,21 我们在急性 IAV H1N1 感染的怀孕 NHP 模型中的初步数据 在 IAV 接种后第 5 天，所有动物均出现肺炎。在怀孕的 NHP 中，流感疾病评分 高于有明显肺外器官损伤（心肌炎、白质损伤）的非妊娠动物。 怀孕的 NHP 在全血和 PBMC 耦合中表现出几乎不存在的早期 Th17 CD4 T 细胞反应 与非怀孕动物相比，在免疫病理学高峰期，肺部的 Th17 细胞显着增加。 肺部和支气管肺泡灌洗液 (BAL) 中的炎症细胞因子和趋化因子在 怀孕动物与非怀孕动物。在目标 1 中，将挑战未怀孕和怀孕的猪尾猕猴 感染 IAV H1N1 A/CA/04/09 或 H3N2 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 Th17 CD4 T 细胞的频率和广谱来确定 IAV 疾病的特异性免疫相关性 先天/适应性免疫反应（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、NLRP3 炎性体激活、CD8 T 细胞）。在目标 2 中，我们将使用批量 和单细胞 RNA 测序，以定义 PBMC、BAL、肺和胎盘内转录组的变化 重点关注 Th17 转录网络和抗病毒先天免疫途径。综上，初步数据 表明怀孕动物中存在异常的 Th17 反应，这对于促进病毒清除和 预防肺损伤。这些研究将是第一个全面分析先天/适应性免疫反应的研究 在急性 IAV 感染期间，以阐明孕妇严重肺部疾病的发病机制。结果来自 这些研究对于 IAV 大流行的防范至关重要，可以测试新型 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.