This early-release report compared licensed influenza antiviral monotherapies and 2-drug combinations in a lethal mouse model of highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b. The objective was to assess whether combining direct-acting antivirals from distinct mechanistic classes improves clinical and virologic outcomes versus single-agent therapy, to inform H5N1 preparedness strategies.
Six- to eight-week-old female BALB/c mice were intranasally challenged with A/mink/Spain/3691–8_22VIR10586–10/2022 at 5 LD50 (90 TCID50 in a 50-µL inoculum). Treatment began at 6 hours postinfection (hpi). Test agents were oseltamivir phosphate (OSP; neuraminidase inhibitor), molnupiravir (MPV; nucleoside analog), and baloxavir acid (BXA; cap-dependent endonuclease inhibitor).
OSP and MPV were administered orally twice daily for 5 days; BXA was given as a single subcutaneous dose. Low-dose groups received OSP 25 mg/kg, MPV 25 mg/kg, or BXA 20 mg/kg; corresponding 2-drug combinations used the same per-drug dosages (OSP/MPV 25/25 mg/kg, BXA/OSP 20/25 mg/kg, BXA/MPV 20/25 mg/kg). Higher-dose comparisons included escalation of OSP and MPV to 50 mg/kg in some groups while maintaining BXA at 20 mg/kg.
Body weight and survival were monitored through 18 days postinfection (dpi) with n = 7 mice per group. Infectious virus titers in lung, brain, and heart were quantified by TCID50 assay at 4 and 6 dpi (n = 3 per time point). Histopathology and influenza A nucleoprotein immunohistochemistry were performed on lung and brain at 6 dpi. Lung RNA sequencing was carried out on samples collected at 6 dpi to evaluate transcriptomic responses.
Vehicle-treated mice exhibited rapid weight loss and uniform mortality. In low-dose monotherapy groups, OSP and MPV each produced partial protection (2 of 7 mice survived), whereas a single BXA dose was more protective (4 of 7 survived) and more consistently limited weight loss.
Dose escalation of monotherapies to 50 mg/kg improved survival outcomes but did not result in complete protection. Across all evaluated 2-drug regimens—including OSP/MPV (25/25 and 50/50 mg/kg), BXA/OSP (20/25 and 20/50 mg/kg), and BXA/MPV (20/25 and 20/50 mg/kg)—combination therapy stabilized body weight and markedly improved survival compared with corresponding monotherapies.
Notably, the BXA-containing combinations showed the greatest protective effect: both tested dose pairs for BXA/OSP and BXA/MPV produced 7/7 survival through 18 dpi. The OSP/MPV combination improved outcomes relative to single agents but did not uniformly produce complete protection in all experiments.
A Bliss-independence analysis of end-of-study (18 dpi) survival produced positive ΔBliss estimates for BXA-containing combinations, most clearly at the 20/25 mg/kg dose pair, consistent with a favorable interaction between agents. At the higher 20/50 mg/kg pairs, ΔBliss values remained positive but were smaller, suggesting an attenuated interaction at greater drug exposure. OSP/MPV combinations showed a weaker, dose-dependent interaction.
Tissue-level virologic control paralleled clinical outcomes. At 4 dpi, BXA and OSP monotherapies—but not MPV monotherapy—reduced lung viral titers, with brain and heart titers near or below the assay limit of detection (LOD), indicating limited early dissemination.
By 6 dpi, vehicle-treated mice had high lung viral loads with detectable virus in brain and heart. BXA monotherapy more consistently kept brain and heart titers at or near the LOD, whereas occasional replication in brain occurred with 25 mg/kg OSP or MPV monotherapy.
Combination therapy further suppressed residual dissemination. At 4 dpi, lung titers in combination-treated groups frequently approached the LOD, most prominently for BXA-containing pairs, and brain and heart titers remained at or near the LOD. By 6 dpi, low lung titers were sustained in combination groups and systemic spread was largely abolished in BXA-containing regimens. OSP/MPV combinations provided intermediate virologic control, reducing pulmonary titers and preventing neuroinvasion and detectable heart virus in many, but not all, instances observed under some monotherapy conditions.
Lung and brain sections examined at 6 dpi were processed for hematoxylin and eosin staining and influenza A nucleoprotein immunohistochemistry. Representative images reported in the study show that combination therapy attenuated histopathologic changes and antigen detection compared with vehicle-treated mice and in several comparisons versus monotherapy groups. The source figures illustrate reduced immunoreactivity and less extensive tissue damage in BXA-containing combination groups relative to vehicle and some monotherapies.
RNA sequencing was performed on lung tissue harvested at 6 dpi to profile treatment-associated effects on host transcriptional responses. Principal component analysis of lung transcriptomic profiles showed separation between treatment groups and suggested attenuation of infection-associated pathway perturbations under combination therapy. The source text reports these analyses and indicates additional pathway-level and differential expression results in the Appendix; detailed transcriptomic pathway findings are reported in the article supplement.
In this lethal mouse model of H5N1 clade 2.3.4.4b infection, combination antiviral therapy improved clinical and virologic outcomes compared with monotherapy. Baloxavir-based combinations (BXA/OSP and BXA/MPV) provided the greatest benefit, achieving complete survival at tested dose pairs, stronger lung viral clearance, and prevention of extrapulmonary dissemination when treatment began 6 hours after intranasal challenge.
The authors conclude that BXA-containing combination regimens warrant consideration in preparedness strategies for severe H5N1 disease because they improved survival, reduced pulmonary viral loads, and prevented systemic spread in this preclinical model.
This article is an early-release version; the Appendix contains additional figures, tables, and detailed analyses. Specifics of some transcriptomic pathway results, full statistical values for all comparisons, and certain experimental subtleties are reported in the Appendix and figures referenced in the source. Where the main text does not provide granular numeric values or full pathways, those details are available in the article supplement per the source. The study used a single mouse strain, a single H5N1 clade 2.3.4.4b isolate, and a defined treatment start time (6 hpi), which may limit direct extrapolation to other exposure routes, hosts, or delayed treatment scenarios described in other H5N1 studies.