Immunopathogenesis of SARS-CoV-2 infection

我们已经编写了一份动物研究提案，该提案已经得到NIAID动物保护和使用委员会和SVC的批准。在BSL3和ABSL3设施为此目的开放后，我们于2020年秋季开始研究。到目前为止，以C57BL/6为背景的WT和huACE2转基因小鼠已经感染了SARS-CoV-2临床分离株。HuACE2小鼠对致命性感染高度敏感，而WT小鼠存活。我们正在交叉小鼠品系，在huACE2背景上测试趋化因子受体基因敲除小鼠，因为人类对Covid病的研究已经报告在CCR9最近的这个区域有强烈的信号，所以优先考虑由CCR簇中的基因编码的受体。我们的目标是首先测试WT LD100剂量，筛选能够提高存活率的受体敲除，因为任何积极的结果都将具有翻译潜力。筛选阴性的受体将在LDzer值重新筛选，以在模型中寻找保护性趋化因子受体。到目前为止，我们已经发现Ccr6基因敲除增加了体重减轻和致命后果的易感性。我们还试图开发一种AAV-huACE2瞬时表达系统，通过避免huACE2转基因/趋化因子受体Ko杂交来加快筛选。我们还在寻找一种SARS-CoV-2毒株，这种毒株已经适应了老鼠的感染，正在加速发现。对于每种类型的实验，将测试四种病毒剂量，以确定是否可以为每种病毒建立无症状、轻度毒力或更高毒力的模型。女性和男性都将接受测试，预计在冠状病毒疾病的临床进展和组织病理学方面存在差异，这是基于小鼠的其他传染病模型，以及在感染SARS-CoV和SARS-CoV-2的人类中观察到的性别差异。在所有随后的实验中，将通过咽拭子、血液和粪便中的病毒恢复；感染前和感染后多个时间间隔的血清抗体效价、血象、血液化学和血清细胞因子；以及感染后一个月的体貌、体重减轻和死亡和存活的进展来评估临床疾病进展。动物最迟在感染后一个月将被二氧化碳和颈椎脱位实施安乐死。在我们的经验中，为了观察存活率的显著差异，我们需要在每个实验中有20只小鼠/队列，由于实验之间存活率的差异，我们需要重复实验3次。这些数字应该足以观察到其他临床数据的显著差异。将需要更少的非感染小鼠，因为预计这些对照之间的差异较小。在本报告期间，我们的KO筛查结果显示了Ackr1缺乏的保护作用以及CCR2、CCR5和Cxcl10缺乏的有害影响。 第二个目标是表征感染SARS-CoV-2的培养人肺外植体的局部免疫反应。我们已经成功地与来自NICHD的L·马戈利斯合作建立了这个系统，并展示了在上皮细胞中的生产性感染。 我们还没有公布这些结果。

We have written an Animal Study Proposal that has been approved by the NIAID Animal Care and Use Committee and the SVC. We began research in fall, 2020 after the BSL3 and ABSL3 facilities opened for this purpose. To date, WT and huACE2 transgenic mice on a C57BL/6 background have been infected with clinical isolates of SARS-CoV-2. The huACE2 mice are highly susceptible to fatal infection whereas the WT mice survive. We are crossing mouse strains to test chemokine receptor knockout mice on the huACE2 background prioritizing receptors encoded by genes in the CCR cluster since human GWAS studies of Covid disease have reported a strong signal in this region most proximal to CCR9. Our goal is to test a WT LD100 dose first, screening for receptor knockouts that increase survival, since any positive results will have translational potential. Receptors that screen negative will be rescreened at an LDzero looking for protective chemokine receptors in the model. To date we have identified Ccr6 knockouts as having increased susceptibility to weight loss and fatal outcome. We are also attempting to develop a AAV-huACE2 transient expression system to accelerate screening by obviating the huACE2 transgenic/chemokine receptor ko crosses. We are also pursuing a strain of SARS-CoV-2 that has been adapted to mouse infection towards accelerating discovery. For each type of experiment four virus doses will be tested to determine if models for asymptomatic, mild virulence or higher virulence can be developed for each virus. Both females and males will be tested and are expected to have differences in clinical progression of coronaviral disease and histopathology, based on other infectious disease models in mice, and differences between the sexes observed in humans infected with SARS-CoV and SARS-CoV-2. In all subsequent experiments, clinical disease progression will be assessed by virus recovery from throat swabs, blood and stool; serum antibody titers, hemograms, blood chemistries and serum cytokines before and at multiple intervals after infection; as well as physical appearance, weight loss, and progression to moribundity and survival for a month after infection. Animals will be euthanized by CO2 and cervical dislocation at the latest one-month post-infection. In our experience, to observe significant differences in survival we will need 20 mice/cohort in each experiment and repeat the experiment 3 times due to the variance in survival between experiments. These numbers should be sufficient to observe significant differences in the other clinical data as well. Fewer non-infected mice will be needed since there is expected to be less variance among these controls. In this reporting period, the results of our ko screen has revealed protective effects of Ackr1 deficiency and harmful effects of Ccr2, Ccr5 and Cxcl10 deficiency. A secondary goal is to characterize the local immune response in cultured human lung explants infected with SARS-CoV-2. We have succeeded in collaboration with L Margolis from NICHD in establishing this system and demonstrating productive infection in epithelial cells. We have not yet published these results.