3 3 5 5 tetramethylbenzidine solution

Mouse serum samples of each group were pooled and heat inactivated at 56 °C for 30 min. Random mouse IgG (Pierce, Waltham, MA, USA) and sera from mock-vaccinated mice were included as negative controls. The three-fold serial dilutions of samples were prepared in a medium containing heat-inactivated FBS. Next, 200 PFU of RSV-HRP were mixed with the diluted mouse sera, incubated for 1 h at 37 °C, and transferred to HEp-2 monolayers. Virus was adsorbed for 1 h and then replaced with fresh medium. Following incubation for 48 h, the supernatant was replaced by 3,3′,5,5′-tetramethylbenzidine solution (Thermo Fisher Scientific, Waltham, MA, USA), incubated for 30 min, and the reaction was stopped by adding 2M sulfuric acid. The optical density at 450 nm was measured. The experiment was performed twice from two independent mouse studies with n = 5 each, with similar results. Error bars represent the standard deviation of the mean of triplicate samples from the pooled sera. Curve-fit analyses were performed using Graphpad Prism 9 and reciprocal titers that achieved 50% neutralization of the RSV-HRP were calculated. A reliable curve fit for RSV-preF^∆CT could not be performed due to the incomplete neutralization. Significances were determined by unpaired two-tailed Student’s t-test with Welch’s correction.

Isolated neutrophils in HBSS buffer or phosphate-buffered saline, pH 7.4 (PBS) were pretreated with vehicle or the indicated concentrations of 4-ABAH, a specific inhibitor of MPO, for 30 min at 37°C. The cells were sonicated in PBS (8 × 10⁶ and 1 × 10⁶/mL for mouse and human neutrophils, respectively) containing protease inhibitor cocktail and 1 mM PMSF. The cell lysate was collected after centrifugation and sample reactions were prepared in duplicates. Each reaction contained the lysates of 4 × 10⁵ mouse neutrophils or 1 × 10⁴ human neutrophils with 0.3 mM H₂O₂ and 10.5% 3,3′,5,5′-Tetramethylbenzidine solution (ThermoFisher). The reaction was quenched with the addition of 0.8 N HCl and the absorbance was read on a PHERAstar plate reader (BMG Labtech) at 450 nm. In some experiments, 1.6 × 10⁸/mL mouse neutrophils were stimulated with or without 10 ng/mL PMA for 10 min at 37°C after pretreatment with vehicle or 500 µM 4-ABAH. The supernatant was collected and used in the MPO activity assay. In other experiments, recombinant human MPO (100 ng) was treated with vehicle or the indicated concentrations of 4-ABAH and used in the assay.