Titres this website by human reference sera and monoclonal antibodies to OMV antigens with known bactericidal titres. Opsonophagocytic activity was measured as respiratory burst [36]. Live meningococci from strain 44/76 and B1723 were used as targets, and human polymorphonuclear leucocytes from

a normal human donor, probed with dihydrorhodamine 123 (Invitrogen, Oslo, Norway), as effector cells. A human serum, different from that used in SBA, served as complement source after passing through a Protein G-column. A positive human reference serum was included as control for complement activity and assay sensitivity. Twofold serial dilutions of the mouse sera were analysed, and respiratory burst

was analysed with a flow cytometer (Partec CyFlow® ML, Partec GmbH, Münster, Germany) gating on the polymorphonuclear population. Opsonic titres were recorded as log2 of the highest reciprocal serum dilution giving ≥50% respiratory burst of the polymorphonuclear leucocytes. Magnetic polystyrene beads (40 mg/ml) (Dynabeads® Talon®; Invitrogen Dynal, Oslo, Norway) were washed with 50 mm Na-phosphate, pH 8.0, 300 mm NaCl and 0.01% Tween-20 according to the manufacturer’s instructions and incubated for 10 min in the same buffer with his-tagged recombinant Omp85. Preliminary experiments showed that 4 mg of beads bound ≤40 μg Omp85, as no Omp85 protein was detected Copanlisib molecular weight after SDS gel electrophoresis of the

supernatant. The recombinant Omp85 protein preparation showed one band in SDS gels of molecular mass about 90 kDa. Due to the small amounts of recombinant Omp85 available, serum pools were used for the adsorption experiments. Differences in antibody levels were analysed with Student’s t-test or Mann–Whitney rank sum test with a SigmaStat 3.1 program (Systat Software, Chicago, IL, USA). Correlations between only SBA and PorA antibody levels were assessed by the non-parametric Spearman’s rank-order correlation test. P-values < 0.05 were considered significant. After induction of transformed meningococci with IPTG, the genetically modified Omp85+ OMVs expressed fivefold higher levels of Omp85 (mean 5.2; range 4.4–7.2 of six determinations) relative to PorA in SDS gels and on blots compared with same levels in the wt 1 and wt 2 OMVs (Fig. 1A,B). The SDS gel also shows that the three OMV preparations contained different levels of the opacity proteins OpcA and OpaJ (Fig. 1A). Omp85+ OMVs expressed negligible levels of both proteins; OpaJ was modestly increased in the wt 2 OMV control, whereas a dominant OpcA band was observed in wt 1 OMVs, as previously reported [33]. These antigens might possibly affect the bactericidal activity of the OMV vaccines. However, OpaJ does not induce bactericidal antibodies in mice [37].

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