Based on the lack of bactericidal activity of the opacity proteins and the similar Omp85 levels in the two wt OMVs, no distinction will be made below between

the wt 1 and wt 2 control see more OMVs. The mice were immunized with the Omp85+ and control wt vaccines as described in Table 1, and their specific Omp85 antibody levels measured by scanning of the Omp85 band intensities on immunoblots (Fig. 2A). The Omp85+ vaccine induced significantly higher Omp85 antibody levels in C57BL/6 (P = 0.023) and OFI mice (P = 0.008) than in Balb/c mice. Whereas all C57BL/6 and OFI mice showed high levels, only half of the Balb/c mice had corresponding responses. Compared with the Omp85+ vaccine, the wt vaccine induced significantly lower Omp85 antibody levels in Balb/c mice (P = 0.035) and C57BL/6 mice (P = 0.001). However, NMRI mice responded to the wt vaccine with antibody levels that were significantly higher than in Balb/c (P = 0.001) and C57BL/6 mice (P = 0.001)

and not significantly different from those in C57BL/6 and OFI mice receiving the Omp85+ vaccine (Fig. 2A). The wt vaccine did not induce significant differences in antibody levels between the Balb/c and C57BL/6 mice, nor did Balb/c AP24534 mouse mice, immunized with the two wt vaccines, show significant differences (data not shown) in support of their similar Omp85 content. Similar results but with lower Omp85 antibody levels were obtained when wt 1 OMVs were used as immunoblotting antigen. The mouse strains displayed no significant differences in PorA antibody levels, Thymidine kinase determined on the same blots, after immunization with the Omp85+ and wt vaccines (Fig. 2B), indicating that the vaccines expressed the same amount of PorA. However, some C57BL/6 mice showed low or no PorA responses with both vaccines. Taken

together, the blotting results indicated that the mice showed distinct strain-dependent antibody responses to Omp85 and PorA. Serum bactericidal assay was performed with strain 44/76 and its derived PorA-negative strain (B1723) as targets (Fig. 3). With strain 44/76, the bactericidal titres induced in Balb/c mice by the Omp85+ and wt vaccines were not significantly different. The same was observed for C57BL/6 mice, implying that the increased Omp85 level, induced by the Omp85+ vaccine, did not contribute to the bactericidal antibodies. However, titres in Balb/c mice, given the Omp85+ vaccine, were slightly higher (P = 0.047) than in C57BL/6 mice. The same trend was also observed with the wt vaccine, but this difference was not significant. The lack of PorA antibody activity in some sera from the C57BL/6 mice, as shown in Fig. 2B, may explain the titre differences. This was supported by the high Spearman’s correlation coefficients between the bactericidal titres and PorA antibody levels for C57BL/6 mice immunized with the Omp85+ (0.734; P = 0.005) and wt vaccine (0.615; P = 0.031).