High throughput profiling of PA amino acid substitutions in an A(H1N1) pdm09 influenza virus that confers resistance to baloxavir in vitro

Abstract

Influenza epidemics and pandemics pose continuous threats to global public health. Current and novel antivirals play an important role in both the prevention and treatment of influenza infection. Baloxavir marboxil (BMX) is a novel pan-influenza antiviral, which was licensed in 2018. Baloxavir acid (BXA), the active form of BMX, inhibits influenza A and B virus replication by directly inhibiting the endonuclease activity of polymerase acid protein (PA) and effectively blocking viral mRNA synthesis. However, resistance to BXA has been reported in clinical settings, which may compromise drug efficacy and limit treatment options. Amino acid substitutions at PA residue 38 (I38F/M/T) that confer reduced susceptibility to BXA in both influenza A and B viruses have been reported from patients after treatment. Identification of mutations other than I38X that confer resistance to BXA is needed to provide guidance for BXA clinical usage. In this study, pools of recombinant A(H1N1)pdm09 viruses with random PA mutations were constructed and selected in the presence of BXA in vitro to identify amino acid substitutions that confer resistance. Pair-end Next Generation Sequencing was applied to profile mutations detected at low frequency. Single round passage experiment allowed selection of variants from the mutagenized virus pool while the serial passage experiment under increasing concentration of BXA allowed both selection and emergence of de novo mutation in the PA terminus domain that confer resistance. Interestingly, single round passage experiment failed to select any known PA mutations that confer resistance to BXA while BXA resistant mutations were selected in the serial passage experiment. After five serial passages under increasing concentration of BXA in vitro, PA-I 38T or PA-I38M mutation were invariably detected from six independently performed experimental replicates, indicating that amino acid substitutions at PA residue 38 may readily emerge upon BXA selection. Additional mutations that were detected at >5% frequency and prior to the emergence of I38T/M mutations during the serial passages were each introduced into the PA protein of recombinant A(H1N1)pdm09 virus for further validation using High-content imaging-based neutralization test and minigenome assay. High-content imaging-based neutralization test confirmed that two novel mutations L106R and E198D showed 16.7 fold and 2.1 fold change in EC50 to BXA, respectively, when compared to the wild-type A(H1N1)pdm09 virus. Minigenome assay also confirmed reduced susceptibility of L106R and E198D mutation to BXA with 12.4 fold and 2.9 fold changes in EC50, respectively. L106R reduced stock virus titer by 2 logs while there was no apparent impact of the E198D mutation on viral yield. In this thesis, two complementary selection methods were performed to maximize the power of identifying mutations confer resistance to BXA. Both known mutations and novel mutations were identified using the serial passage method. The newly identified PA mutations may provide reference for monitoring BXA sensitivity of influenza viruses after treatment and in surveillance studies. The results support the use of deep-mutational scanning strategy to identify novel mutations that may confer resistance to antiviral agents.