Virological and clinical outcomes in outpatients treated with baloxavir or neuraminidase inhibitors for A(H3N2) influenza: A multicenter study of the 2022-2023 season.

While clinical trials have illuminated both the virological and clinical efficacy of baloxavir for influenza and post-treatment viral resistance, these aspects warrant further study in real-world settings. In response, we executed a prospective, observational study of the Japanese 2022-2023 influenza season. A cohort of 73 A(H3N2)-diagnosed outpatients-36 treated with baloxavir, 20 with oseltamivir, and 17 with other neuraminidase inhibitors (NAIs)-were analyzed. Viral samples were collected before and after administering an antiviral on days 1, 5, and 10, respectively. Cultured viruses were amplified using RT-PCR and sequenced to detect mutations. Fever and other symptoms were tracked via self-reporting diaries. In the baloxavir cohort, viral detection was 11.1% (4/36) and 0% (0/36) on day 5 and day 10, respectively. Two isolates from day 5 (5.6%, 2/36) manifested I38T/M-substitutions in the polymerase acidic protein (PA). For oseltamivir and other NAIs, viral detection rates were 60.0% (12/20) and 52.9% (9/17) on day 5, and 16.7% (3/18) and 6.3% (1/16) on day 10, respectively. No oseltamivir-resistant neuraminidase mutations were identified after treatment. Median fever durations for the baloxavir, oseltamivir, and other NAI cohorts were 27.0, 38.0, and 36.0 h, respectively, with no significant difference. Two patients harboring PA I38T/M-substitutions did not exhibit prolonged fever or other symptoms. These findings affirm baloxavir's virological and clinical effectiveness against A(H3N2) in the 2022-2023 season and suggest limited clinical influence of post-treatment resistance emergence.

Clinical outcomes of baloxavir versus oseltamivir in immunocompromised patients.

BACKGROUND: Neuraminidase inhibitors, including oseltamivir, are the treatment standard for influenza. Baloxavir, a novel antiviral, demonstrated comparable outcomes to oseltamivir in outpatients with influenza. Baloxavir was equally effective as oseltamivir in a retrospective study of hospitalized patients with influenza at our institution. However, the efficacy of baloxavir in immunocompromised patients is unclear. METHODS: We conducted a retrospective cohort study of immunocompromised adult patients hospitalized with influenza A who received baloxavir from January 2019 to April 2019 or oseltamivir from January 2018 to April 2018. Demographic and clinical outcomes were assessed. Primary outcomes were time from antiviral initiation to resolution of hypoxia and fever. Secondary outcomes were length of stay (LOS), intensive care unit (ICU) care, ICU LOS, and 30-day mortality. RESULTS: Of 95 total patients, 52 received baloxavir and 43 received oseltamivir. Other than younger age (57.5 vs. 65; p = .035) and longer duration between vaccination and symptom onset (114 vs. 86 days; p = .001) in the baloxavir group, baseline characteristics did not differ. H1 was the predominant subtype in the baloxavir group (65.3%) versus H3 in the oseltamivir group (85.7%). When comparing baloxavir to oseltamivir, there was no significant difference in median time from antiviral initiation to resolution of hypoxia (59.9 vs. 42.5 h) and to resolution of fever (21.6 vs. 26.6 h). There were no differences in secondary outcomes. CONCLUSION: Baloxavir was not associated with longer time to resolution of hypoxia or fever in comparison to oseltamivir. Results must be taken in context of variations in seasonal influenza subtype and resistance rates.

A protectin DX (PDX) analog with in vitro activity against influenza A(H1N1) viruses.

Antiviral therapy based on neuraminidase (oseltamivir) or polymerase (baloxavir marboxil) inhibitors plays an important role in the management of influenza infections. However, the emergence of drug resistance and the uncontrolled inflammatory response are major limitations in the treatment of severe influenza disease. Protectins D1 (PD1) and DX (PDX), part of a family of pro-resolving mediators, have previously demonstrated anti-influenza activity as well as anti-inflammatory properties in various clinical contexts. Herein, we synthetized a series of simplified PDX analogs and assessed their in vitro antiviral activity against influenza A(H1N1) viruses, including oseltamivir- and baloxavir-resistant variants. In ST6GalI-MDCK cells, the PDX analog AN-137B reduced viral replication in a dose-dependent manner with IC50 values of 23.8 for A/Puerto Rico/8/1934 (H1N1) and between 32.6 and 36.7 µM for susceptible and resistant A(H1N1)pdm09 viruses. In MTS-based cell viability experiments, AN-137B showed a 50% cellular cytotoxicity (CC50 ) of 638.7 µM with a resulting selectivity index of 26.8. Of greater importance, the combination of AN-137B with oseltamivir or baloxavir resulted in synergistic and additive in vitro effects, respectively. Treatment of lipopolysaccharide (LPS)-stimulated macrophages with AN-137B resulted in a decrease of iNOS activity as shown by the reduction of nitrite production, suggesting an anti-inflammatory effect. In conclusion, our results indicate that the protectin analog AN-137B constitutes an interesting therapeutic modality against influenza A virus, warranting further evaluation in animal models.

An orally active entry inhibitor of influenza A viruses protects mice and synergizes with oseltamivir and baloxavir marboxil.

Seasonal or pandemic illness caused by influenza A viruses (IAVs) is a major public health concern due to the high morbidity and notable mortality. Although there are several approved drugs targeting different mechanisms, the emergence of drug resistance calls for new drug candidates that can be used alone or in combinations. Small-molecule IAV entry inhibitor, ING-1466, binds to hemagglutinin (HA) and blocks HA-mediated viral infection. Here, we show that this inhibitor demonstrates preventive and therapeutic effects in a mouse model of IAV with substantial improvement in the survival rate. When administered orally it elicits a therapeutic effect in mice, even after the well-established infection. Moreover, the combination of ING-1466 with oseltamivir phosphate or baloxavir marboxil enhances the therapeutic effect in a synergistic manner. Overall, ING-1466 has excellent oral bioavailability and in vitro absorption, distribution, metabolism, excretion, and toxicity profile, suggesting that it can be developed for monotherapy or combination therapy for the treatment of IAV infections.

Repositioning baloxavir marboxil as VISTA agonist that ameliorates experimental asthma.

V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA), a novel negative checkpoint regulator, plays an essential role in allergic pulmonary inflammation in mice. Treatment with a VISTA agonistic antibody could significantly improve asthma symptoms. Thus, for allergic asthma treatment, VISTA targeting may be a compelling approach. In this study, we examined the functional mechanism of VISTA in allergic pulmonary inflammation and screened the FDA-approved drugs for VISTA agonists. By using mass cytometry (CyTOF), we found that VISTA deficiency primarily increased lung macrophage infiltration in the OVA-induced asthma model, accompanied by an increased proportion of M1 macrophages (CD11b+F4/80+CD86+) and a decreased proportion of M2 macrophages (CD11b+F4/80+CD206+). Further in vitro studies showed that VISTA deficiency promoted M1 polarization and inhibited M2 polarization of bone marrow-derived macrophages (BMDMs). Importantly, we discovered baloxavir marboxil (BXM) as a VISTA agonist by virtual screening of FDA-approved drugs. The surface plasmon resonance (SPR) assays revealed that BXM (KD = 1.07 µM) as well as its active form, baloxavir acid (BXA) (KD = 0.21 µM), could directly bind to VISTA with high affinity. Notably, treatment with BXM significantly ameliorated asthma symptoms, including less lung inflammation, mucus secretion, and the generation of Th2 cytokines (IL-5, IL-13, and IL-4), which were dramatically attenuated by anti-VISTA monoclonal antibody treatment. BXM administration also reduced the pulmonary infiltration of M1 macrophages and raised M2 macrophages. Collectively, our study indicates that VISTA regulates pulmonary inflammation in allergic asthma by regulating macrophage polarization and baloxavir marboxil, and an old drug might be a new treatment for allergic asthma through targeting VISTA.

Discovery of a Macrocyclic Influenza Cap-Dependent Endonuclease Inhibitor.

Influenza viruses (IFVs) have caused several pandemics and have claimed numerous lives since their first record in the early 20th century. While the outbreak of COVID-19 seemed to expel influenza from the sight of people for a short period of time, it is not surprising that it will recirculate around the globe after the coronavirus has mutated into a less fatal variant. Baloxavir marboxil (1), the prodrug of baloxavir (2) and a cap-dependent endonuclease (CEN) inhibitor, were approved by the FDA for the first treatment in almost 20 years. Despite their high antiviral potency, drug-resistant variants have been observed in clinical trials. Herein, we report a novel CEN inhibitor 8 with a delicately designed macrocyclic scaffold that exhibits a significantly smaller shift of inhibitory activity toward baloxavir-resistant variants.

Structural Studies of Inhibitors with Clinically Relevant Influenza Endonuclease Variants.

Vital to the treatment of influenza is the use of antivirals such as Oseltamivir (Tamiflu) and Zanamivir (Relenza); however, antiviral resistance is becoming an increasing problem for these therapeutics. The RNA-dependent RNA polymerase acidic N-terminal (PAN) endonuclease, a critical component of influenza viral replication machinery, is an antiviral target that was recently validated with the approval of Baloxavir Marboxil (BXM). Despite its clinical success, BXM has demonstrated susceptibility to resistance mutations, specifically the I38T, E23K, and A36 V mutants of PAN. To better understand the effects of these mutations on BXM resistance and improve the design of more robust therapeutics, this study examines key differences in protein-inhibitor interactions with two inhibitors and the I38T, E23K, and A36 V mutants. Differences in inhibitor binding were evaluated by measuring changes in binding to PAN using two biophysical methods. The binding mode of two distinct inhibitors was determined crystallographically with both wild-type and mutant forms of PAN. Collectively, these studies give some insight into the mechanism of antiviral resistance of these mutants.

The impact of PA/I38 substitutions and PA polymorphisms on the susceptibility of zoonotic influenza A viruses to baloxavir.

Genetic reassortment of avian, swine, and human influenza A viruses (IAVs) poses potential pandemic risks. Surveillance is important for influenza pandemic preparedness, but the susceptibility of zoonotic IAVs to the cap-dependent endonuclease inhibitor baloxavir acid (BXA) has not been thoroughly researched. Although an amino acid substitution at position 38 in the polymerase acidic protein (PA/I38) in seasonal IAVs reduces BXA susceptibility, PA polymorphisms at position 38 are rarely seen in zoonotic IAVs. Here, we examined the impact of PA/I38 substitutions on the BXA susceptibility of recombinant A(H5N1) viruses. PA mutants that harbored I38T, F, and M were 48.2-, 24.0-, and 15.5-fold less susceptible, respectively, to BXA than wild-type A(H5N1) but were susceptible to the neuraminidase inhibitor oseltamivir acid and the RNA polymerase inhibitor favipiravir. PA mutants exhibited significantly impaired replicative fitness in Madin-Darby canine kidney cells at 24 h postinfection. In addition, in order to investigate new genetic markers for BXA susceptibility, we screened geographically and temporally distinct IAVs isolated worldwide from birds and pigs. The results showed that BXA exhibited antiviral activity against avian and swine viruses with similar levels to seasonal isolates. All viruses tested in the study lacked the PA/I38 substitution and were susceptible to BXA. Isolates harboring amino acid polymorphisms at positions 20, 24, and 37, which have been implicated in the binding of BXA to the PA endonuclease domain, were also susceptible to BXA. These results suggest that monitoring of the PA/I38 substitution in animal-derived influenza viruses is important for preparedness against zoonotic influenza virus outbreaks.

Comparison of clinical efficacy and safety of baloxavir marboxil versus oseltamivir as the treatment for influenza virus infections: A systematic review and meta-analysis.

INTRODUCTION: Baloxavir marboxil (BXM), a newly developed cap-dependent endonuclease inhibitor, is widely used to treat influenza virus infections in inpatients and outpatients. A previous meta-analysis included only outpatients and patients suspected of having an influenza virus infection based on clinical symptoms. However, whether BXM or oseltamivir is safer and more effective for inpatients remains controversial. Therefore, we conducted a systematic review and meta-analysis validating the effectiveness and safety of BXM versus oseltamivir in inpatients with influenza virus. METHODS: The Scopus, EMBASE, PubMed, Ichushi, and CINAHL databases were systematically searched for articles published until January 2023. The outcomes were mortality, hospitalization period, incidence of BXM- or oseltamivir-related adverse events, illness duration, and changes of virus titers and viral RNA load in patients with influenza virus infections. RESULTS: Two randomized controlled trials with 1624 outpatients and two retrospective studies with 874 inpatients were enrolled. No deaths occurred in outpatients treated with BXM or oseltamivir. Among inpatients, BXM reduced mortality (p = 0.06) and significantly shortened hospitalization period (p = 0.01) compared to oseltamivir. In outpatients, BXM had a significantly lower incidence of adverse events (p = 0.03), reductions in influenza virus titers (p < 0.001) and viral RNA loads (p < 0.001), and a tendency to be a shorter illness duration compared with that of oseltamivir (p = 0.27). CONCLUSIONS: Our meta-analysis showed that BXM was safer and more effective in patients than oseltamivir; thus, supporting the use of BXM for the initial treatment of patients with proven influenza virus infection.

Baloxavir marboxil use for critical human infection of avian influenza A H5N6 virus.

BACKGROUND: Recent outbreaks of avian influenza and ongoing virus reassortment have drawn focus on spill-over infections. The increase in human infections with highly pathogenic avian influenza H5N6 virus and its high fatality rate posed a potential threat, necessitating the search for a more effective treatment. METHODS: Longitudinal clinical data and specimens were collected from five H5N6 patients after admission. All patients received antiviral treatment of either sequential monotherapy of oseltamivir and baloxavir or the two drugs in combination. Severity of illness; viral load in sputum, urine, and blood; and cytokine levels in serum and sputum were serially analyzed. FINDINGS: All patients developed acute respiratory distress syndrome (ARDS) and viral sepsis within 1 week after disease onset. When delayed oseltamivir showed poor effects, baloxavir was administered and rapidly decreased viral load. In addition, levels of IL-18, M-CSF, IL-6, and HGF in sputum and Mig and IL-18 in serum that reflected ARDS and sepsis deterioration, respectively, were also reduced with baloxavir usage. However, three patients eventually died from exacerbation of underlying disease and secondary bacterial infection. Nonsurvivors had more severe extrapulmonary organ dysfunction and insufficient H5N6 virus-specific antibody response. CONCLUSIONS: For critical human cases of H5N6 infection, baloxavir demonstrated effects on viral load and pulmonary/extrapulmonary cytokines, even though treatment was delayed. Baloxavir could be regarded as a first-line treatment to limit continued viral propagation, with potential future application in avian influenza human infections and poultry workers exhibiting influenza-like illness. FUNDING: This work was funded by the National Natural Science Foundation of China (81761128014).

Early Fever Resolution in Early Childhood Influenza Treated with Baloxavir Marboxil: A Retrospective Study Compared to Those with Oseltamivir.

Background and Objectives: Baloxavir marboxil is a novel cap-dependent endonuclease inhibitor prescribed for influenza treatment. Unlike neuraminidase inhibitors like oseltamivir, which impair viral release from infected host cells, baloxavir blocks influenza virus proliferation by inhibiting viral mRNA transcription. This study aimed to compare the effectiveness of baloxavir and oseltamivir for the treatment of early childhood influenza. Materials and Methods: Of 1410 patients diagnosed with influenza between 2015 and 2018 at a Japanese primary care outpatient clinic, 1111 pediatric patients aged 0-6 years who were treated with baloxavir (n = 555) or oseltamivir (n = 556) were enrolled retrospectively. The following clinical factors were compared between patients treated with baloxavir and oseltamivir: age, sex, time from fever onset to drug administration (<24 h or 24-48 h), time from drug administration to fever reduction, influenza type (A or B), and influenza vaccination before disease onset. The duration of the fever, which was used as an index of clinical effectiveness, was compared using the log-rank test. Clinical factors associated with fever duration were determined using multivariate logistic regression analysis. Results: Median age (3.0 vs. 2.5 years), influenza type A (99% vs. 47%), median duration from drug administration to fever resolution (1 day vs. 2 days), and influenza vaccination (done, 41% vs. not done, 65%) were significantly different between the baloxavir and oseltamivir groups (p < 0.001). The number of patients with a fever duration of one day was 553 (99.6%) in the baloxavir group and 6 (1.1%) in the oseltamivir group (p < 0.001). Baloxavir use was only significantly associated with fever duration in the multivariate analysis (odds ratio 50,201, p < 0.001). Apparent adverse effects were not observed in the baloxavir-treated group. Conclusions: Baloxavir treatment resulted in a shorter fever duration than oseltamivir treatment in early childhood influenza.

Baloxavir safety and clinical and virologic outcomes in influenza virus-infected pediatric patients by age group: age-based pooled analysis of two pediatric studies conducted in Japan.

BACKGROUND: Anti-influenza treatment is important for children and is recommended in many countries. This study assessed safety, clinical, and virologic outcomes of baloxavir marboxil (baloxavir) treatment in children based on age and influenza virus type/subtype. METHODS: This was a post hoc pooled analysis of two open-label non-controlled studies of a single weight-based oral dose of baloxavir (day 1) in influenza virus-infected Japanese patients aged < 6 years (n = 56) and ≥ 6 to < 12 years (n = 81). Safety, time to illness alleviation (TTIA), time to resolution of fever (TTRF), recurrence of influenza illness symptoms and fever (after day 4), virus titer, and outcomes by polymerase acidic protein variants at position I38 (PA/I38X) were evaluated. RESULTS: Adverse events were reported in 39.0 and 39.5% of patients < 6 years and ≥ 6 to < 12 years, respectively. Median (95% confidence interval) TTIA was 43.2 (36.3-68.4) and 45.4 (38.9-61.0) hours, and TTRF was 32.2 (26.8-37.8) and 20.7 (19.2-23.8) hours, for patients < 6 years and ≥ 6 to < 12 years, respectively. Symptom and fever recurrence was more common in patients < 6 years with influenza B (54.5 and 50.0%, respectively) compared with older patients (0 and 25.0%, respectively). Virus titers declined (day 2) for both age groups. Transient virus titer increase and PA/I38X-variants were more common for patients < 6 years. CONCLUSIONS: The safety and effectiveness of single-dose baloxavir were observed in children across all age groups and influenza virus types. Higher rates of fever recurrence and transient virus titer increase were observed in children < 6 years. TRIAL REGISTRATION: Japan Pharmaceutical Information Center Clinical Trials Information JapicCTI-163,417 (registered 02 November 2016) and JapicCTI-173,811 (registered 15 December 2017).

Chemical scaffold recycling: Structure-guided conversion of an HIV integrase inhibitor into a potent influenza virus RNA-dependent RNA polymerase inhibitor designed to minimize resistance potential.

Influenza is one of the leading causes of disease-related mortalities worldwide. Several strategies have been implemented during the past decades to hinder the replication cycle of influenza viruses, all of which have resulted in the emergence of resistant virus strains. The most recent example is baloxavir marboxil, where a single mutation in the active site of the target endonuclease domain of the RNA-dependent-RNA polymerase renders the recent FDA approved compound ∼1000-fold less effective. Raltegravir is a first-in-class HIV inhibitor that shows modest activity to the endonuclease. Here, we have used structure-guided approaches to create rationally designed derivative molecules that efficiently engage the endonuclease active site. The design strategy was driven by our previously published structures of endonuclease-substrate complexes, which allowed us to target functionally conserved residues and reduce the likelihood of resistance mutations. We succeeded in developing low nanomolar equipotent inhibitors of both wild-type and baloxavir-resistant endonuclease. We also developed macrocyclic versions of these inhibitors that engage the active site in the same manner as their 'open' counterparts but with reduced affinity. Structural analyses provide clear avenues for how to increase the affinity of these cyclic compounds.

Baloxavir Marboxil Shows Anomalous Conversion of Crystal Forms from Stable to Metastable through Formation of Specific Solvate Form.

Baloxavir marboxil is a novel cap-dependent endonuclease inhibitor of influenza. This study aimed to identify its polymorphs and their relationship with crystal engineering. Polymorph screening by evaporation gave forms I-III and solvate forms IV and V. Heating enabled the conversion of form III to form II, but did not enable that of forms I and II. The solvent-mediated transformation of the forms I-III by magnetic stirring in various solvents resulted in the formation of form I. These results indicate that form I is the stable form. However, all crystal forms transformed to form II after magnetic stirring in a 50% acetonitrile aqueous solution, which was not obtained from water or acetonitrile. The suspension in a 50% acetonitrile aqueous solution exhibited a novel X-ray diffraction pattern as shown in form VI. The measurement of the suspension by solid-state 13C-nuclear magnetic resonance revealed that the spectra of forms II and VI were similar. From these results, we conclude that the drug forms a solvate with both water and acetonitrile and spontaneously transforms to form II upon rapid desolvation under ambient conditions. This study elucidates the mechanism of unexpected convergence to a metastable form in a specific solvent and contributes to the crystal engineering of baloxavir marboxil.

Effect of E23G/K, F36V, N37T, E119D, and E199G polymerase acidic protein substitutions on the replication and baloxavir susceptibility of influenza B viruses.

Baloxavir marboxil (baloxavir) is a highly effective, single-dose influenza therapeutic. Identification of molecular markers in the target polymerase acidic (PA) protein that can diminish baloxavir efficacy is an ongoing goal of the scientific community. In this study, we generated recombinant Victoria-lineage and Yamagata-lineage influenza B viruses (IBVs) containing 6 substitutions (E23G/K, F36V, N37T, E119D, and E199G) spanning the endonuclease domain of the PA. Although 5 of these PA substitutions negatively impacted in vitro polymerase activity and replication kinetics, particularly in the Victoria-lineage IBV background, viruses with E119D exhibited activity levels comparable to those of wild-type viruses. Moreover, only E119D moderately decreased the susceptibility of IBVs to baloxavir (resulting in ∼2.0-fold to 2.6-fold elevated EC50s); viruses with the other substitutions exhibited normal drug inhibition. These results show that E119D may reduce the baloxavir susceptibility of IBVs without compromising their replicative fitness. Overall, this study expands the molecular landscape of PA substitutions potentially affecting baloxavir efficacy against IBV.

Genetic Evolution of Avian Influenza A (H9N2) Viruses Isolated from Domestic Poultry in Uganda Reveals Evidence of Mammalian Host Adaptation, Increased Virulence and Reduced Sensitivity to Baloxavir.

A (H9N2) avian influenza A viruses were first detected in Uganda in 2017 and have since established themselves in live bird markets. The aim of this study was to establish the subsequent genetic evolution of H9N2 viruses in Uganda. Cloacal samples collected from live bird market stalls in Kampala from 2017 to 2019 were screened by RT-PCR for influenza A virus and H9N2 viruses were isolated in embryonated eggs. One hundred and fifty H9N2 isolates were subjected to whole genome sequencing on the Illumina MiSeq platform. The sequence data analysis and comparison with contemporary isolates revealed that the virus was first introduced into Uganda in 2014 from ancestors in the Middle East. There has since been an increase in nucleotide substitutions and reassortments among the viruses within and between live bird markets, leading to variations in phylogeny of the different segments, although overall diversity remained low. The isolates had several mutations such as HA-Q226L and NS-I106M that enable mammalian host adaptation, NP-M105V, PB1-D3V, and M1-T215A known for increased virulence/pathogenicity and replication, and PA-E199D, NS-P42S, and M2-S31N that promote drug resistance. The PA-E199D substitution in particular confers resistance to the endonuclease inhibitor Baloxavir acid, which is one of the new anti-influenza drugs. Higher EC50 was observed in isolates with a double F105L+E199D substitution that may suggest a possible synergistic effect. These H9N2 viruses have established an endemic situation in live bird markets in Uganda because of poor biosecurity practices and therefore pose a zoonotic threat. Regular surveillance is necessary to further generate the needed evidence for effective control strategies and to minimize the threats.

Effect of Baloxavir and Oseltamivir in Combination on Infection with Influenza Viruses with PA/I38T or PA/E23K Substitutions in the Ferret Model.

Amino acid substitutions I38T and E23K in the influenza polymerase acidic (PA) protein lead to reduced susceptibility to the influenza antiviral drug baloxavir. The in vivo effectiveness of baloxavir and oseltamivir for treatment of these viruses is currently unknown. Using patient-derived influenza isolates, combination therapy was equally effective as monotherapy in reducing viral titers in the upper respiratory tract of ferrets infected with A(H1N1pdm09)-PA/E23K or A(H3N2)-PA/I38T. When treated with baloxavir plus oseltamivir, infection with a mixture of PA/I38T or PA/E23K and corresponding wild-type virus was characterized by a lower selection of viruses with reduced baloxavir susceptibility over the course of infection compared to baloxavir monotherapy. De novo emergence of the oseltamivir resistance mutation NA/H275Y occurred in ferrets treated with oseltamivir alone but not in ferrets treated with baloxavir plus oseltamivir. Our data suggest that combination therapy with influenza drugs with different mechanisms of action decreased the selection pressure for viruses with reduced drug susceptibility. IMPORTANCE Influenza viruses cause significant morbidity and mortality worldwide but can be treated with antiviral drugs. In 2018, a highly effective antiviral drug, baloxavir marboxil, was licensed. However, the selection of viruses with baloxavir resistance was relatively high following treatment, which may compromise the effectiveness of the drug. Here, we took two different influenza viruses that are resistant to baloxavir and tested the effectiveness alone and in combination with oseltamivir (a second influenza antiviral drug) in the ferret model. Our findings suggest that combination treatment may be a more effective method than monotherapy to reduce the selection of resistant viruses. These results may have important clinical implications for the treatment of influenza.

Clinical effectiveness of baloxavir marboxil against influenza in three seasons.

BACKGROUND: Previous reports have not clarified the difference in clinical efficacy between baloxavir and oseltamivir against influenza. METHODS: A prospective observational study was performed during 2017-2018, 2018-2019, and 2019-2020 influenza seasons. The primary endpoint of this study was to compare the duration of fever between patients who received baloxavir and those who received oseltamivir. RESULTS: A total of 235 influenza-infected patients (3-18 years of age), including 91 who received oseltamivir and 144 who received baloxavir, were enrolled. The proportions of influenza A(H3N2) virus, influenza A(H1N1)pdm09 virus, and influenza B virus-infected patients were 31.5%, 42.6%, and 26.0%, respectively. Patients who received oseltamivir were significantly younger than those who received baloxavir. Univariate analyses showed that the duration of fever was shorter with baloxavir than with oseltamivir against influenza virus overall, influenza A virus, influenza B virus, and influenza A(H1N1)pdm09 virus, but not for influenza A(H3N2) virus. In multivariate analyses, hazard ratios for influenza virus overall (0.53 [95% CI, 0.38-0.73]), influenza B virus (0.16 [95% CI, 0.07-0.41]), and influenza A(H1N1)pdm09 virus (0.55 [95% CI, 0.32-0.93]) were significantly lower in the patients who received baloxavir than those who received oseltamivir. However, the differences between influenza A virus and influenza A(H3N2) virus were not significant between the two groups. CONCLUSION: For influenza virus overall, influenza B virus, and influenza A(H1N1)pdm09 virus, baloxavir treatment resulted in shorter duration of fever than oseltamivir treatment, but not for influenza A virus and influenza A(H3N2) virus.

Influenza A virus polymerase acidic protein E23G/K substitutions weaken key baloxavir drug-binding contacts with minimal impact on replication and transmission.

Baloxavir marboxil (BXM) is approved for treating uncomplicated influenza. The active metabolite baloxavir acid (BXA) inhibits cap-dependent endonuclease activity of the influenza virus polymerase acidic protein (PA), which is necessary for viral transcription. Treatment-emergent E23G or E23K (E23G/K) PA substitutions have been implicated in reduced BXA susceptibility, but their effect on virus fitness and transmissibility, their synergism with other BXA resistance markers, and the mechanisms of resistance have been insufficiently studied. Accordingly, we generated point mutants of circulating seasonal influenza A(H1N1)pdm09 and A(H3N2) viruses carrying E23G/K substitutions. Both substitutions caused 2- to 13-fold increases in the BXA EC50. EC50s were higher with E23K than with E23G and increased dramatically (138- to 446-fold) when these substitutions were combined with PA I38T, the dominant BXA resistance marker. E23G/K-substituted viruses exhibited slightly impaired replication in MDCK and Calu-3 cells, which was more pronounced with E23K. In ferret transmission experiments, all viruses transmitted to direct-contact and airborne-transmission animals, with only E23K+I38T viruses failing to infect 100% of animals by airborne transmission. E23G/K genotypes were predominantly stable during transmission events and through five passages in vitro. Thermostable PA-BXA interactions were weakened by E23G/K substitutions and further weakened when combined with I38T. In silico modeling indicated this was caused by E23G/K altering the placement of functionally important Tyr24 in the endonuclease domain, potentially decreasing BXA binding but at some cost to the virus. These data implicate E23G/K, alone or combined with I38T, as important markers of reduced BXM susceptibility, and such mutants could emerge and/or transmit among humans.

Effects of baloxavir and oseltamivir antiviral therapy on the transmission of seasonal influenza in China: A mathematical modeling analysis.

New antiviral influenza treatments can effectively alleviate illness while reducing viral shedding. However, how such effects can translate into lower population infections of seasonal influenza in China remains unknown. To shed light on the public health impacts of novel antiviral agents for influenza, we constructed a dynamic transmission model to simulate the seasonal influenza epidemics in China. Two antivirus treatments, baloxavir and oseltamivir, were evaluated by estimating their impacts on the incidences of influenza infection in a single flu season. In the base-case analysis of a 10% antiviral treatment uptake rate, 2760 and 3420 per 10 000 persons contracted influenza under the treatment of baloxavir and oseltamivir, respectively. These incidence rates amounted to an 18.90% relative risk reduction (RRR) of infection associated with baloxavir in relation to oseltamivir. The corresponding RRR was 82.16% when the antiviral treatment uptake rate was increased to 35%. In addition, the peak of the prevalence of infected individuals per 10 000 persons under the baloxavir treatment was 177 (range: 93-274) fewer than that of oseltamivir. Our analyses suggest that the baloxavir treatment strategy reduces the incidence of influenza in China compared with oseltamivir in the setting of a seasonal flu epidemic. Also, increasing the uptake rate of antiviral treatment can potentially prevent millions of infections during a single flu season.