Impact of Baloxavir Resistance-Associated Substitutions on Influenza Virus Growth and Drug Susceptibility

Abstract

Baloxavir marboxil (baloxavir) is a recently FDA-approved influenza virus polymerase acidic (PA) endonuclease inhibitor. Several PA substitutions have been demonstrated to confer reduced susceptibility to baloxavir; however, their impacts on measurements of antiviral drug susceptibility and replication capacity when present as a fraction of the viral population have not been established. We generated recombinant A/California/04/09 (H1N1)-like viruses (IAV) with PA I38L, I38T, or E199D substitutions and B/Victoria/504/2000-like virus (IBV) with PA I38T. These substitutions reduced baloxavir susceptibility by 15.3-, 72.3-, 5.4-, and 54.5-fold, respectively, when tested in normal human bronchial epithelial (NHBE) cells. We then assessed the replication kinetics, polymerase activity, and baloxavir susceptibility of the wild-type:mutant (WT:MUT) virus mixtures in NHBE cells. The percentage of MUT relative to WT virus necessary to detect reduced baloxavir susceptibility in phenotypic assays ranged from 10% (IBV I38T) to 92% (IAV E199D). While I38T did not alter IAV replication kinetics or polymerase activity, IAV PA I38L and E199D MUTs and the IBV PA I38T MUT exhibited reduced replication levels and significantly altered polymerase activity. Differences in replication were detectable when the MUTs comprised ≥90%, ≥90%, or ≥75% of the population, respectively. Droplet digital PCR (ddPCR) and next-generation sequencing (NGS) analyses showed that WT viruses generally outcompeted the respective MUTs after multiple replication cycles and serial passaging in NHBE cells when initial mixtures contained ≥50% of the WT viruses; however, we also identified potential compensatory substitutions (IAV PA D394N and IBV PA E329G) that emerged and appeared to improve the replication capacity of baloxavir-resistant virus in cell culture. IMPORTANCE Baloxavir marboxil, an influenza virus polymerase acidic endonuclease inhibitor, represents a recently approved new class of influenza antivirals. Treatment-emergent resistance to baloxavir has been observed in clinical trials, and the potential spread of resistant variants could diminish baloxavir effectiveness. Here, we report the impact of the proportion of drug-resistant subpopulations on the ability to detect resistance in clinical isolates and the impact of substitutions on viral replication of mixtures containing both drug-sensitive and drug-resistant variants. We also show that ddPCR and NGS methods can be successfully used for detection of resistant subpopulations in clinical isolates and to quantify their relative abundance. Taken together, our data shed light on the potential impact of baloxavir-resistant I38T/L and E199D substitutions on baloxavir susceptibility and other biological properties of influenza virus and the ability to detect resistance in phenotypic and genotypic assays.

Keywords: baloxavir resistance; influenza; polymerase substitutions.

FIG 1

Replication kinetics of the IAV (A and B) WT:MUT I38L, (C and D) WT:MUT I38T, (E and F) WT:MUT E199D, and (G and H) IBV WT:MUT I38T virus mixtures in NHBE cells. Cells were infected with the virus mixtures at an MOI of 0.01 for 1 h before being washed, overlaid with fresh medium, and incubated at 37°C (IAV) or 33°C (IBV). At 24, 48, and 72 hpi, supernatants were collected and titrated by plaque assay in MDCK cells. Fold changes in the area under the curve for the WT:MUT mixtures compared to the WT virus are shown in parentheses. *, P < 0.05, and °, P < 0.01, compared to the values for the respective WT virus by one-way ANOVA.

FIG 2

Changes in the WT:MUT ratios after multiple replication cycles in NHBE cells. RNA was extracted from supernatants and analyzed either by ddPCR (A, C, E, and G) or NGS (B, D, F, and H). The dashed line represents the percentage of the MUT virus needed to increase the EC₅₀ value by 3-fold compared to the respective WT virus as determined by virus yield reduction assay in NHBE cells (Table 4). Red shading indicates abundance of the MUT virus resulting in EC₅₀ values higher than 3-fold the EC₅₀ of the respective WT virus (3EC₅₀); gray shading indicates abundance of the MUT virus resulting in EC₅₀ values lower than 3EC₅₀ of the respective WT virus as determined by virus yield reduction assay.

FIG 3

Changes in the WT:MUT ratios after serial passaging in NHBE cells. RNA was extracted from supernatants and analyzed by either ddPCR (A, C, E, and G) or NGS (B, D, F, and H). The dashed line represents the percentage of the MUT virus needed to increase the EC₅₀ value by 3-fold compared to the respective WT virus as determined by virus yield reduction assay in NHBE cells (Table 4). Red shading indicates abundance of the MUT virus resulting in EC₅₀ values higher than 3-fold the EC₅₀ of the respective WT virus (3EC₅₀); gray shading indicates abundance of the MUT virus resulting in EC₅₀ values lower than 3EC₅₀ of the respective WT virus as determined by virus yield reduction assay.

FIG 4

Susceptibility measurements of IAV (A) WT:MUT I38L, (B) WT:MUT I38T, (C) WT:MUT E199D, and (D) IBV WT:MUT I38T virus mixtures to baloxavir by plaque reduction assay in MDCK-SIAT1 cells. Cells were infected with influenza virus for 1 h at 37°C (IAV) or 33°C (IBV). The cells were then washed, overlaid with minimal essential medium containing 0.25% agarose and baloxavir, and incubated at 37°C (IAV) or 33°C (IBV) for 2 or 3 days, respectively. Cells were stained with 0.1% crystal violet in 10% formaldehyde solution, and the plaques were counted. The concentration of baloxavir that caused a 50% decrease in the PFU titer compared to control wells without drug was defined as the EC₅₀. The results of two independent experiments were averaged. IAV I38L, I38T, and E199D, and IBV I38T MUTs exhibited EC₅₀ value fold changes of 10.3-, 32.4-, 2.7-, and 5.0-fold when present at 100%, respectively, compared to the respective WTs. The dashed line represents the percentage of the MUT virus needed to increase the EC₅₀ value by 3-fold compared to the respective WT virus (Table 4). Red shading indicates abundance of the MUT virus resulting in EC₅₀ values higher than 3-fold the EC₅₀ of the respective WT virus (3EC₅₀); gray shading indicates abundance of the MUT virus resulting in EC₅₀ values lower than 3EC₅₀ of the respective WT virus. *, P < 0.05, and °, P < 0.01, compared to the values for the respective WT virus by one-way ANOVA.

FIG 5

Susceptibility of IAV RNPs containing PA (A) I38L, (B) I38T, (C) E199D, and (D) IBV RNPs containing PA I38T to baloxavir. HEK 293T cells were treated with baloxavir for 1 h before being transfected with a mixture of PB1, PB2, PA (WT:MUT ratio [percentage]), and NP plasmids. The cells were the incubated at 37°C (IAV) or 33°C (IBV) for 24 h. Cell extracts were lysed, and luciferase levels were assayed with a dual-luciferase-based assay. The concentration of baloxavir that caused a 50% reduction in RNP activity compared to untreated cells was defined as the IC₅₀. Experiments were performed at least in triplicate. IAV I38L, I38T, and E199D, and IBV I38T MUT RNPs exhibited IC₅₀ value fold changes of 5.3-, 139.5-, 2.7-, and 25.7-fold when present at 100%, respectively, compared to the respective WT RNPs. The dashed line represents the percentage of the MUT RNP needed to increase the IC₅₀ value by 3-fold compared to the respective WT RNP (3IC₅₀) (Table 4). Red shading indicates abundance of the MUT RNP resulting in IC₅₀ values higher than IC₅₀ of the respective WT RNP; gray shading indicates abundance of the MUT RNP resulting in EC₅₀ values lower than 3IC₅₀ of the respective WT virus. *, P < 0.05, and °, P < 0.01, compared to the values for the respective WT RNP by one-way ANOVA.

FIG 6

Susceptibility of IAV (A) WT:MUT I38L, (B) WT:MUT I38T, (C) WT:MUT E199D, and (D) IBV WT:MUT I38T virus mixtures to baloxavir by virus yield reduction assay in NHBE cells. Cells were pretreated with baloxavir for 2 h before being infected with WT:MUT virus mixtures (MOI of 0.01 PFU/cell). After 1 h, the cells were washed, baloxavir-containing basal medium was replaced, and cells were incubated at 37°C (IAV) or 33°C (IBV) for 2 days. Viral titers in the collected supernatants were determined by plaque assay in MDCK cells. The concentration of baloxavir that caused a 50% decrease in the PFU titer compared to control wells without drug was defined as the EC₅₀. The results of two independent experiments were averaged. IAV I38L, I38T, and E199D, and IBV I38T MUTs exhibited EC₅₀ value fold changes of 15.3-, 72.3-, 5.4-, and 54.5-fold when present at 100%, respectively, compared to the respective WTs. The dashed line represents the percentage of the MUT virus needed to increase the EC₅₀ value by 3-fold compared to the respective WT virus (3EC₅₀) (Table 4). Red shading indicates abundance of the MUT virus resulting in EC₅₀ values higher than 3EC₅₀ of the respective WT virus; gray shading indicates abundance of the MUT virus resulting in EC₅₀ values lower than 3EC₅₀ of the respective WT virus. *, P < 0.05, and °, P < 0.01, compared to the values for the respective WT virus by one-way ANOVA.

FIG 7

Abundance of the MUT virus in the supernatants collected after virus yield reduction assay in NHBE cells as measured by ddPCR. The dashed line represents the percentage of the MUT virus needed to increase the EC₅₀ value by 3-fold compared to the respective WT virus as determined by virus yield reduction assay in NHBE cells (3EC₅₀) (Table 4). Red shading indicates abundance of the MUT virus resulting in EC₅₀ values higher than 3EC₅₀ of the respective WT virus; gray shading indicates abundance of the MUT virus resulting in EC₅₀ values lower than 3EC₅₀ of the respective WT virus as determined by virus yield reduction assay.