Comprehensive assessment of amino acid substitutions in the trimeric RNA polymerase complex of influenza A virus detected in clinical trials of baloxavir marboxil

Abstract Background Baloxavir marboxil (BXM) is an approved drug that selectively targets cap‐dependent endonuclease on PA subunit in the RNA polymerase complex of influenza A and B viruses. Amino acid substitutions at position 38 in the PA subunit were identified as a major pathway for reduced susceptibility to baloxavir acid (BXA), the active form of BXM. Additionally, substitutions found at positions E23, A37, and E199 in the PA subunit impact BXA susceptibility by less than 10‐fold. Methods We comprehensively evaluated the impact of novel amino acid substitutions identified in PA, PB1, and PB2 subunits in BXM clinical trials and influenza sequence databases by means of drug susceptibility and replicative capacity. Results PA/I38N in A(H1N1)pdm09 and PA/I38R in A(H3N2) were newly identified as treatment‐emergent substitutions in the CAPSTONE‐2 study. The I38N substitution conferred reduced susceptibility by 24‐fold, whereas replicative capacity of the I38N‐substituted virus was impaired compared with the wild‐type. The I38R‐substituted virus was not viable in cell culture. All other mutations assessed in this extensive study did not significantly affect BXA susceptibility (< 2.4‐fold change). Conclusion These results provide additional information on the impact of amino acid substitutions in the trimeric viral polymerase complex to BXA susceptibility and will further support influenza surveillance.


| INTRODUC TI ON
Influenza is an acute infectious disease caused by the influenza virus, and the worldwide epidemics each year result in approximately 3-5 million seriously ill cases and approximately 290 000-650 000 deaths. 1 Antiviral treatment is recommended for the management of influenza infections, particularly in high-risk individuals such as elderly and immunocompromised persons. Neuraminidase inhibitors (NAIs: oseltamivir, zanamivir, peramivir) are widely used as the current treatment for influenza, 2 while adamantanes, M2 ion channel inhibitors, are no longer used due to widespread resistance in circulating influenza viruses. 3,4 While NAI-resistant A(H1N1)pdm09 viruses are currently only detected at a frequency of <1% among circulating viruses, 2 community clusters of such variant viruses have been detected, [5][6][7] emphasizing the need for antivirals with a novel mechanism of action.
Baloxavir marboxil (BXM) became available for the treatment of uncomplicated influenza in otherwise healthy and high-risk patients in a number of countries, following its approval in Japan and the United States in 2018. 8,9 Baloxavir acid (BXA), the active form of BXM, selectively and potently blocks a catalytic center of cap-dependent endonuclease (CEN) located in the polymerase acid (PA) protein of the influenza polymerase complex, which consists of PA, polymerase basic 1 (PB1), and PB2 subunits. 10,11 The CEN is highly conserved across all types of influenza viruses 12 and plays an essential role in the transcription, protein synthesis, and viral genome replication, 13 and therefore, BXA displays broad-spectrum activity against influenza A, B, C, and D viruses. 14,15 In clinical trials, single-dose BXM treatment was superior to placebo in relieving influenza symptoms and, additionally, superior to both oseltamivir and placebo in reducing the viral load. 8,9 However, amino acid (AA) substitutions at position I38 (T/M/F) in the PA subunit have been identified as the most common treatment-emergent substitutions associated with reduced susceptibility to BXA. 11,16 Influenza surveillance studies conducted in Japan during the 2018-19 influenza season confirmed treatment-emergence of PA/I38T and PA/I38M variants in A(H3N2)-infected subjects. 17,18 A(H1N1)pdm09 and A(H3N2) viruses harboring PA/I38T substitution were detected in some few subjects without prior BXM treatment, suggesting the possibility of human-to-human transmission of the variant viruses. 18,19 In addition to the I38 substitutions, E23K/G, A37T, and E199G substitutions were identified in the PA subunit that affect BXA susceptibility by less than 10-fold. 11,20,21 Therefore, consecutive monitoring of variant viruses with reduced BXA susceptibility is required to identify new potential genetic markers for the purpose of influenza surveillance.
It has been well demonstrated that mutations in NA conferring resistance to NAIs can negatively impact the viral replicative capacity, but additional HA mutations can also compensate these fitness cost. 22 K229R in the PB1 subunit of influenza A viruses confers resistance to the viral RNA polymerase inhibitor favipiravir, and the fitness cost caused by this mutation can be compensated by a P653L substitution in PA that restores the fitness while maintaining favipiravir resistance. 23 Therefore, AA substitutions located at distal position from drug-binding sites may impact drug sensitivity or compensate impaired fitness.
Here, we report phenotypic analyses of AA substitutions in PA, PB1, and PB2 subunits, which were detected in clinical trials and influenza surveillance. This additional information on BXA susceptibility and replicative capacity of viruses with these substitutions will further support influenza surveillance.

| Genotypic analyses of clinical samples
In the clinical trials, Sanger sequencing was conducted using paired pre-and post-treatment nasopharyngeal/pharyngeal swab samples from BXM-treated subjects to identify treatment-emergent AA substitutions. In the CAPSTONE-2 study, next-generation sequencing (NGS) was also employed with the samples meeting the following criteria: (a) subjects shedding A(H3N2) viruses with PA/I38T substitution, (b) virology data (viral titer and RNA) are available at days 1, 2, 3 or 4, and 5 or 6, and (c) virus rebound was detected, defined as >1.5 log 10 TCID 50 /mL increases in viral titer from previous adjacent time point. NGS was conducted as reported previously, and a threshold frequency of >1% was adopted for calling variant viruses. 16

| Phenotypic analyses of variant viruses
The plaque reduction assay was conducted as described previously. Evaluation of virus-replicative capacity was previously described. 11 Briefly, 2 × 10 5 cells/well MDCK or 1 × 10 6 cells/well RPMI2650 cells were seeded on 24-well plates 1 day prior to infection. MDCK and RPMI2650 cells were infected with 10 and 100 TCID 50 /well of the viruses, respectively. The infected cells were incubated at 37°C in a 5% CO 2 incubator for 1 hour, followed by exchanging the inoculum to MEM containing 3 μg/mL trypsin and incubation at 37°C in the 5% CO 2 incubator. The culture supernatants were collected at the indicated time points, and viral titers (log 10 T-CID 50 /mL) were determined on MDCK cells.

| Assessment of novel PA/I38X substitutions detected in clinical trials
Resistance monitoring in phase 2 (T0821) and pediatric (T0822 [Japic CTI-163417]) trials revealed treatment-emergent I38T/F/M substitutions in PA, which confer reduced susceptibility to BXA (Table 1 and Table S1). 11 In order to assess treatment-emergent  In order to assess the impact of these detected I38 substitutions on BXA susceptibility, the recombinant A(H1N1) and A(H3N2) viruses harboring the individual substitution were generated and subjected to susceptibility testing. Plaque reduction assay revealed that the A(H1N1) viruses with polymorphic I38V and L substitutions displayed reduced BXA susceptibility by 2-fold and 6-fold, respectively (Table 1), consistent with a previous report. 26 The replicative capacities of the A(H1N1) and A(H3N2) viruses with I38V and L substitutions were comparable to those of the wild-type viruses in canine MDCK and human RPMI2650 cells (Figure 1 and Figure S1).
Viruses bearing the I38N and I38S substitutions showed reduced BXA susceptibility by 24-fold and 12-fold, respectively (Table 1), but also, the recombinant A(H1N1) and A(H3N2) viruses with I38N and I38S substitutions exhibited significant fitness cost in MDCK and RPMI2650 cells (Figure 1 and Figure S1). The I38R virus could not be obtained by reverse genetics, suggesting I38R conferred severe growth defect to the virus.

| Assessment of non-I38 PA substitutions and PB1/2 substitutions detected in clinical trials
Impact of PA substitutions at other positions than I38 PA on BXA susceptibility was also assessed. Results for AA substitutions identified in T0821 and T0822 clinical trials were previously reported. 11 None of the newly tested AA substitutions identified in the clinical trials T0831 and T0832 did significantly impact BXA susceptibility (< 3-fold change in EC 50 ) (Table S1) (Table S1).

| Assessment of PA and PB2 substitutions identified in extended analyses of clinical trial data
Amino acid substitutions at positions associated with baloxavir resistance were identified from NCBI database influenza sequences and from extended analyses of virologic data from clinical trials (Table S2) (Table S2). substitutions identified in the clinical trial were compensatory mutations. The replicative capacity was previously evaluated for substitutions A20S + I38F and I38T + E623K, 11 and I38T + S60P and I38T + I201T were tested in this study ( Figure S2). However, these substitutions were unable to restore the growth impairment of the  Writing-review & editing (lead).

DATA AVA I L A B I L I T Y S TAT E M E N T
All relevant data are within the manuscript and its Supporting Information.