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 documented 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 11 patients given Remdesivir and 47 patients not given Remdesivir. Patient recruitment is ongoing with the goal of enrolling an additional 50 patients. 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 and treatment course. Given the potential dual impact of Remdesivir on polymerase and exonuclease activities, it is possible that Remdesivir enhances viral diversity, and so these studies will be complemented with deep sequencing studies 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 over the course of severe disease and will determine the impact of Remdesivir treatment on that variation. In Aim 2, in vitro oligonucleotide extension assays and ex vivo mini-replicon 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 discovered mutations 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 at a few concentrations near the EC50. The phylodynamic histories of impactful mutants 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.
|Effective start/end date||7/1/21 → 6/30/23|
- National Institute of Allergy and Infectious Diseases (1R21AI163912-01)
Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.