Generation of immune sera and SBA.

Eight female BALB/c mice (6 to 8 weeks old; Charles River, Margate, UK) were immunized with recombinant fHbp (20 μg) adsorbed to aluminum hydroxide [final composition, 0.5 mg/ml Al(OH)₃, 10 mM histidine-HCl] by mixing overnight at 4°C or with Bexsero (total protein, 20 μg). Antigens were given by the intraperitoneal route on days 0, 21, and 35, and sera were collected on day 49 by terminal anesthesia and cardiac puncture. All animal experiments were carried out under protocols reviewed and approved by the Home Office, United Kingdom, under license number PPL 30/3194.

To measure SBA, N. cinerea was grown overnight on BHI agar prior to replating onto solid medium and was grown for a further 5 h at 37°C in 5% CO₂. A total of 5 × 10⁴ CFU/ml of N. cinerea was mixed with an equal volume of baby rabbit complement (Cedarlane) diluted 1:10 in SBA buffer (Dulbecco's PBS containing 0.1% [wt/vol] glucose, 1 mM CaCl₂, 0.5 mM MgCl₂). Sera from individual mice were pooled and heat inactivated for 1 h at 56°C prior to being added to wells in 2-fold dilutions starting at 1:8. Control wells contained either no serum or no complement. Following incubation for 1 h at 37°C, 10 μl from each well was plated onto BHI agar in triplicate, and the number of surviving bacteria was determined after overnight growth. The SBA was expressed as the reciprocal of the highest dilution of serum required to kill more than 50% of the bacteria relative to a no-serum control (49).