A Synergistic Dual-Antibody Cocktail Targeting α-Toxin Protects Against Invasive Staphylococcus aureus Infection by Neutralizing Virulence and Enhancing Host Defense.

Staphylococcus aureus is a leading cause of severe invasive infections and is associated with high mortality. The pore-forming α-toxin (Hla) is a key virulence determinant that drives disease severity. We identified two high-affinity anti-Hla monoclonal antibodies, Hm0399 and Hm0411, derived from the sera of volunteers vaccinated with the S. aureus vaccine rFSAV. Mechanistic analyses-including toxin neutralization assays, structure solution, and effector-function characterization-revealed that although both antibodies recognize overlapping epitopes on Hla, they confer protection through distinct and complementary mechanisms. Hm0411 potently neutralizes Hla toxicity by blocking receptor engagement through a defined salt-bridge network, whereas Hm0399 engages a hydrogen-bond interface that enhances Fc-mediated effector functions. A dual-antibody cocktail comprising Hm0399 and Hm0411 (Hm3-4) demonstrated robust therapeutic efficacy in the USA300 S. aureus sepsis model and pneumonia model. Notably, protection was not compromised by pre-existing immune imprinting. Transcriptomic profiling indicated enhanced Fcγ receptor-mediated phagocytosis and suppression of pro-inflammatory Ca2+ signaling pathways. In addition, combining Hm3-4 with ultra-low doses of vancomycin or linezolid synergistically improved therapeutic outcomes in experimental sepsis. Collectively, these findings establish a dual-antibody strategy targeting Hla that integrates virulence neutralization with augmentation of host immune defense, providing a framework for next-generation immunotherapies against invasive S. aureus infection.