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Introduction Influenza viruses continue to pose a global health threat, and available antiviral therapies are limited by resistance and reduced efficacy. Host-directed drugs such as MEK inhibitors have emerged as promising alternatives. Zapnometinib, a clinical-stage MEK inhibitor, has shown both antiviral and immunomodulatory activity. However, its impact on antigen presentation at the level of the HLA-I ligandome has not been investigated. Methods Label-free LC–MS/MS was applied to analyze HLA-I–presented peptides in human lung adenocarcinoma Calu-3 cells. Cells were infected with Influenza A virus (IAV) H3N2/Fukui and treated with zapnometinib. Surface HLA-I expression was quantified by flow cytometry, and immunopeptidomic analyses were applied to assess ligandome alterations and functional pathway enrichment. Results Zapnometinib treatment and IAV infection did not significantly alter HLA-I surface expression (fold changes 1.01-1.13, p 0.05). Immunopeptidomics revealed allotype-specific changes in the relative abundance of HLA-I-presented peptides (approximately 3–12% change per allotype), and statistically significant modulation of defined ligand subsets (about 3–14% of ligands per condition; log 2 fold change ≥ 2, adjusted p 0.05). Functional annotation analysis showed condition-specific enrichment in distinct cellular pathways, such as interferon-induced pathways, including IFI16 upregulation during infection and its downregulation upon MEK inhibition, indicating selective modulation of antiviral responses, cell cycle and RNA-processing pathways under zapnometinib treatment, and adhesion-related pathways under combined treatment. Ligandome remodeling after IAV H3N2/Fukui infection strongly affected antiviral hubs (IFIH1, DHX58, IFI16), whereas zapnometinib caused no consistent or significant reduction. Discussion/conclusion This study provides the first evidence that zapnometinib and IAV H3N2/Fukui induce remarkable effects on the HLA-I ligandome plasticity without substantially affecting overall HLA-I expression. These findings highlight the dual role of MEK inhibitors in modulating both viral replication and immune recognition through pathway- and allotype-specific changes in peptide presentation rather than bulk HLA-I levels. Our results support further investigation of zapnometinib as a host-directed antiviral strategy with the potential to contribute to targeted immunomodulation in antiviral therapy.
Hamza et al. (Tue,) studied this question.