Immunopathogenesis of SARS-CoV-2 infection

我们已经编写了一份动物研究提案，该提案已获得NIAID动物护理和使用委员会和SVC的批准。 在BSL 3和ABSL 3设施为此目的开放后，我们于2020年秋季开始研究。迄今为止，C57 BL/6背景下的WT和huACE 2转基因小鼠已感染SARS-CoV-2的临床分离株。 huACE 2小鼠对致命性感染高度易感，而WT小鼠存活。 我们正在交叉小鼠品系以测试huACE 2背景下的趋化因子受体敲除小鼠，优先考虑CCR簇中基因编码的受体，因为Covid疾病的人类GWAS研究报告了最接近CCR 9的该区域的强信号。 我们的目标是首先测试WT LD 100剂量，筛选增加存活率的受体敲除，因为任何阳性结果都具有翻译潜力。筛选阴性的受体将在LD零下重新筛选，以寻找模型中的保护性趋化因子受体。 到目前为止，我们已经确定Ccr 6基因敲除增加了对体重减轻和致命结局的易感性。 我们还试图开发AAV-huACE 2瞬时表达系统，以通过避免huACE 2转基因/趋化因子受体ko杂交来加速筛选。 我们还在寻找一种适应小鼠感染的SARS-CoV-2菌株，以加速发现。 对于每种类型的实验，将检测四种病毒剂量，以确定是否可以为每种病毒开发无症状、轻度毒力或更高毒力的模型。 将对雌性和雄性动物进行检测，基于小鼠中的其他传染病模型，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，并且在感染SARS-CoV和SARS-CoV-2的人类中观察到性别差异。 在所有随后的实验中，将通过从咽拭子、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滴度、血象、血液化学和血清细胞因子;以及身体外观、体重减轻和向濒死的进展和感染后一个月的存活率来评估临床疾病进展。 最迟在感染后1个月，通过CO2和颈椎脱位对动物实施安乐死。 根据我们的经验，为了观察存活率的显著差异，我们将在每个实验中需要20只小鼠/群组，并且由于实验之间的存活率的差异而重复实验3次。 这些数字应足以观察到其他临床数据的显著差异。 将需要较少的未感染小鼠，因为预期这些对照之间的差异较小。 在本报告期内，我们的ko筛选结果显示Ackr 1缺乏的保护作用和Ccr 2、Ccr 5和Cxcl 10缺乏的有害作用。 第二个目标是表征感染SARS-CoV-2的培养人肺外植体中的局部免疫应答。 我们已经成功地与来自NICHD的L Margolis合作建立了这个系统，并证明了上皮细胞中的生产性感染。 我们尚未公布这些结果。

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.