2, 1 and 5 μg doses based on total protein. Two other groups of mice received 5 μg of GMMA from the Double KO (lpxL1, gna33 KO) OE fHbp mutant or 5 μg GMMA from the Triple KO mutant strain. Control mice were immunised with 5 μg recombinant fHbp ID1 or aluminium hydroxide only. All vaccines were adsorbed on 3 mg/mL Aluminium hydroxide in a 100 μL formulation containing 10 mM Histidine and 0.9 mg/mL
NaCl. Sera were SP600125 supplier stored at −80 °C until use. All animal work was approved by the Italian Animal Ethics Committee (AEC project number 14112011). Anti-fHbp IgG antibody titres were measured by ELISA as previously described . The coating antigen was 1 μg/mL non-lipidated recombinant hexa-Histidine-tagged fHbp ID1 . Serial five-fold dilutions of the serum samples starting at 1:100 were analysed. Secondary antibody was a 1:2000 dilution of alkaline phosphatase-conjugated goat-anti mouse IgG (Invitrogen, cat, no 62-6522, Lot 437983A). The titre was defined as the extrapolated dilution resulting in absorption of 1 at 405 nm after 30 min of incubation with 1 mg/mL 4-nitrophenyl phosphate disodium salt hexahydrate (Sigma–Aldrich) diluted in 1 M diethanolamine and 0.5 mM MgCl2, pH 9.8. Serum bactericidal antibody (SBA) activities were measured as described before . Bacteria were incubated
at 37 °C, 5% CO2 in Mueller–Hinton broth containing 0.25% glucose and 0.02 mM Cytidine-5′-monophospho-N-acetylneuraminic acid sodium salt (Sigma–Aldrich). The cells were washed with Dulbecco’s PBS buffer (Sigma–Aldrich) containing 1% BSA. Each selleck chemicals reaction mixture contained approximately 400 colony-forming units, 20% human complement screened for lack of bactericidal activity against the target strain and serial dilutions of the serum second samples starting at 1:10. Bactericidal titres were defined as the reciprocal extrapolated dilution resulting in 50% killing of bacteria after 60 min incubation at 37 °C compared to the mean number of bacteria in five control reactions
at time 0. For statistical analysis, antibody titres were log 10 transformed. ELISA titres <100 were assigned the value 50, SBA titres <10 were assigned the value 5. Mann–Whitney U test was used to compare pairs of values. A probability value of <0.05 was considered statistically significant. The analysis was performed with the Graph Pad Prism software 5.01. Nine group W strains (six carrier and three case isolates) with PorA subtype P1.5,2, collected in Ghana between 2003 and 2007, were screened as candidate GMMA production strains. To identify the isolate with highest GMMA production, gna33 was deleted from all strains. In some isolates, simultaneous deletion of the capsule decreased the GMMA release compared to the gna33 single knock-out (KO). Therefore, we generated gna33 and capsule double KO mutants of the nine W strains and compared GMMA production. These double-mutant strains released two to five-fold higher amounts of GMMA than a representative group W wild type strain ( Fig. 1A).