Lactoferrin blocks orthopoxvirus entry via heparan sulphate and regulates host antiviral pathways.

Current antiviral therapies for orthopoxviruses face critical challenges, including limited efficacy and significant toxicity, which impede outbreak containment and clinical management. Here, we identify lactoferrin as a potent antiviral agent against multiple orthopoxvirus strains. Combination administration of lactoferrin with brincidofovir or tecovirimat demonstrated additive efficacy, suggesting a potential clinical strategy to reduce individual drug toxicity. Mechanistically, lactoferrin blocks viral entry by competitively binding to heparan sulphate proteoglycans (HSPGs). It also suppresses viral replication by regulating host antiviral pathways, including down-regulating cytokines and upregulating TGF-β-dependent antiviral signalling pathways. We identify TGFBI as a virus-responsive target regulated by lactoferrin. Lactoferrin treatment restores TGFBI expression and activates downstream MAPK/ERK and JAK2/STAT3 signalling cascades, leading to enhanced interferon production and interferon-stimulated gene (ISG) expression. In a murine vaccinia virus (VACV) infection model, lactoferrin treatment reduced lung viral loads and histological damage. These results underscore lactoferrin's distinctive dual antiviral mechanism and highlight its translational potential as a safe and cost-effective prophylactic or therapeutic agent. It is particularly beneficial for immunocompromised populations in resource-limited settings during orthopoxvirus outbreaks.