Assessing the Risk of SARS-CoV-2 Remdesivir Resistance

项目总结 SARS-CoV-2大流行在全球的迅速蔓延导致了一项紧急和持续的评估 《2019年冠状病毒病治疗战略汇编》(新冠肺炎)。而几个主持人- 正在使用定向疗法治疗严重疾病，包括单抗和 地塞米松，只有一种紧急使用授权的抗病毒药物，核苷酸类似物需要 (GS-5734)。雷米西韦被认为通过抑制病毒依赖RNA的RNA聚合酶Nsp12， 核苷酸模拟和早期链终止。随着雷米希韦治疗新冠肺炎的使用升级，它 对于理解产生抗病毒耐药性的性质和风险至关重要。我们假设主机内部 Nsp12基因的变异可能导致雷米昔韦耐药性的进化。在这项试探性拨款中，我们建议 通过量化住院患者随时间出现的SARS-CoV-2病毒多样性来检验这一假设 在接受和不接受瑞希韦治疗的情况下(目标1)，并通过确定已知的Nsp12突变对 聚合酶活性和对瑞昔维韦的敏感性(目标2)。在目标1中，我们将纵向利用生物库 采集西北纪念医院新冠肺炎住院患者的鼻咽拭子。 在登记后每4天收集一次标本，最多一个月，包括8名患者的标本 鉴于雷米德韦尔。我们正在继续收集标本，并打算再招募50名新冠肺炎患者 在接下来的几个月。来自每个样本的SARS-CoV-2病毒的深度全基因组测序将 对每个患者体内检测到的随时间推移的病毒变异进行分类。变化的性质和程度 将根据病历数据确定的时间点、病毒载量和治疗过程进行比较。这些 研究将补充对体外产生的病毒进行深度测序，这些病毒存在于不同的 雷米西韦的浓度。总体而言，这项工作将确定SARS-CoV-2宿主内的性质和范围 在住院患者中发生的变异，并将决定雷米昔韦治疗对其的影响 变种。在目标2中，将使用体外寡核苷酸延伸分析来评估先前 描述了Nsp12突变对聚合酶活性和雷米西韦疗效的影响。记录在案的突变附近 Redesivir结合口袋和Nsp12活性位点，新发现的单倍型出现在我们的患者群体中， 而被发现处于选择压力下的Nsp12突变株将首先进行聚合酶活性评估，然后进行 因为她的敏感度。有影响力的突变和单倍型的系统动力学历史将被 在种群中寻找选择的证据。总而言之，这些结果将评估突发事件的风险 此外，还可以识别潜在的耐药突变，以便进行积极监测。

PROJECT SUMMARY The rapid spread of the SARS-CoV-2 pandemic around the globe has led to an urgent and ongoing evaluation of therapeutic strategies for the treatment of Coronavirus Disease 2019 (COVID-19). While a handful of host- directed therapies are being used for the treatment of severe disease, including monoclonal antibodies and dexamethasone, there is only one antiviral with emergency use authorization, the nucleotide analog remdesivir (GS-5734). Remdesivir is thought to inhibit the viral RNA-dependent RNA polymerase, Nsp12, through nucleotide mimicry and early chain termination. As use of remdesivir for the treatment of COVID-19 escalates, it is essential to understand the nature and risk of developing antiviral resistance. We hypothesize that intra-host variation in Nsp12 could allow for the evolution of remdesivir resistance. In this exploratory grant, we propose to test this hypothesis by quantifying the SARS-CoV-2 viral diversity that arises in hospitalized patients over time with and without remdesivir treatment (Aim 1), and by determining the impact of known Nsp12 mutations on polymerase activity and sensitivity to remdesivir (Aim 2). In Aim 1, we will leverage a biobank of longitudinally collected nasopharyngeal swabs from hospitalized COVID-19 patients at Northwestern Memorial Hospital. Specimens were collected every 4 days after enrollment for up to one month, including specimens from 8 patients given remdesivir. We are continuing to collect specimens and intend to enroll an additional 50 COVID-19 patients over the coming months. Deep, whole genome sequencing of SARS-CoV-2 viruses from each specimen will be performed to classify the viral variants detected within each patient over time. The nature and extent of variation will be compared by time point, viral load, and treatment course as determined from medical record data. These studies will be complemented with deep sequencing of viruses produced ex vivo in the presence of varying concentrations of remdesivir. Overall, this work will determine the nature and extent of SARS-CoV-2 intra-host variation that develops in hospitalized patients and will determine the impact of remdesivir treatment on that variation. In Aim 2, in vitro oligonucleotide extension assays will be used to assess the impact of previously described Nsp12 mutations on polymerase activity and remdesivir efficacy. Documented mutations near the remdesivir binding pocket and Nsp12 active site, newly documented haplotypes arising in our patient population, and Nsp12 mutants found to be under selective pressure will be assessed first for polymerase activity followed by remdesivir sensitivity. The phylodynamic histories of impactful mutations and haplotypes will be followed to look for evidence of selection in the population. Together, these results will assess the risk of emergent remdesivir resistance and additionally identify potential resistance mutations for active monitoring.