Source Data for Enhanced Staphylococcus aureus protection by uncoupling the a-toxin-ADAM10 interaction during murine neonatal vaccination. Tomaszewski KL, Blanchard M et al. Nature Communications. https://doi.org/10.1038/s41467-024-52714-7

Experiments in this dataset include: standard ELISAs and toxin neutralization assays for serological analysis; flow and mass cytometry for T and B cell analysis, in-vitro and cell-based assays for characterization of Hla antigens and skin infection modeling with S. aureus for antigen-adjuvant efficacy. For antibody evaluation, a fixed concentration of purified alpha-toxin was incubated with serially diluted sera samples (immunized mice or from hospitalized human patients) and a standard HRP-based ELISA or a toxin neutralization assay with rabbit red blood cells was performed for antibody titers. For Hla variant functional analysis, the hemolytic activity was evaluated by incubating serially diluted toxin concentrations with rabbit red blood cells, supernatants were read at 450nm. Hla variants were analyzed for binding and oligomerization capability with [35S] labeled Hla incubated on rabbit red blood cells and analyzed by liquid scintillation and SDS-gels visualized using a phosphor imager. The stability of each toxin variant was measured by quantifying the melting temperature of each toxin variant (50% denaturation point) using Sypro Orange and analyzed from 20 to 90 °C in increments of 0.3°C. For S. aureus challenge and immunological analysis, C57BL/6 mice 6 weeks of age were anesthetized via intraperitoneal injection of ketamine (20 mg/kg) and xylazine (5 mg/kg) followed by right flank subcutaneous challenge with 1x108 CFU S. aureus strain USA300/LAC in 50µL PBS. Draining lymph nodes were collected either after immunization and/or infection and single cell suspensions at 1x10^6 were fluorescent labeled for extracellular and intracellular antibodies to analyze the T and B cell compartments via spectral flow cytometry. Single cell suspensions of 3x10^6 cells were also cisplatin labeled and stained for cell surface markers and intracellular antigens of interest and analyzed by CyTOF mass cytometry.Detailed experiment methodology used in this study is fully described in the Methods section of Enhanced Staphylococcus aureus protection by uncoupling the a-toxin-ADAM10 interaction during murine neonatal vaccination. Tomaszewski KL, Blanchard M et al. Nature Communications.