Ex vivo model of meningococcal bacteremia using human blood for measuring vaccine-induced serum passive protective activity

Abstract

The binding of complement factor H (fH) to meningococci was recently found to be specific for human fH. Therefore, passive protective antibody activity measured in animal models of meningococcal bacteremia may overestimate protection in humans, since in the absence of bound fH, complement activation is not downregulated. We developed an ex vivo model of meningococcal bacteremia using nonimmune human blood to measure the passive protective activity of stored sera from 36 adults who had been immunized with an investigational meningococcal multicomponent recombinant protein vaccine. Before immunization, human complement-mediated serum bactericidal activity (SBA) titers of > or = 1:4 against group B strains H44/76, NZ98/254, and S3032 were present in 19, 11, and 8% of subjects, respectively; these proportions increased to 97, 22, and 36%, respectively, 1 month after dose 3 (P < 0.01 for H44/76 and S3032). Against the two SBA-resistant strains, NZ98/254 and S3032, passive protective titers of > or = 1:4 were present in 11 and 42% of sera before immunization, respectively, and these proportions increased to 61 and 94% after immunization (P < 0.001 for each strain). Most of the sera with SBA titers of <1:4 and passive protective activity showed a level of killing in the whole-blood assay (>1 to 2 log(10) decreases in CFU/ml during a 90-min incubation) similar to that of sera with SBA titers of > or = 1:4. In conclusion, passive protective activity was 2.6- to 2.8-fold more frequent than SBA after immunization. The ability of SBA-negative sera to kill Neisseria meningitidis in human blood where fH is bound to the bacteria provides further evidence that SBA titers of > or = 1:4 measured with human complement may underestimate meningococcal immunity.

FIG. 1.

Serum PP activity in a human ex vivo model of meningococcal bacteremia. N. meningitidis strains NZ98/254 and S3032 were incubated with human blood from the donor whose serum was used as the complement source for measuring serum SBA and OPA, together with a 1:4 dilution of heat-inactivated pre- or postimmunization serum from a subject whose SBA titers were <1:4 against both strains. With the preimmunization serum (solid lines), the CFU/ml of both strains were unchanged during the 90-min incubation period, whereas in the presence of the postimmunization serum (dotted line), there were >2 log₁₀ decreases in the respective CFU/ml from those at time zero.

FIG. 2.

Reproducibility of measurements of PP activity in sera from individual subjects. (A) Strain NZ98/254; (B) strain S3032. PP was measured in three independent assays, performed over 3 months on test sera diluted 1:4. Data from the first 14 subjects are shown, represented by three data points (X) per subject for each serum sample. Serum samples are labeled with the subject number followed by the letter A for preimmunization sera and B for postimmunization sera.

FIG. 3.

PP activity against meningococcal bacteremia in an ex vivo model using human blood. The results are shown for individual pre- and postimmunization sera from the 36 adults immunized with three doses of the meningococcal recombinant protein vaccine. Each symbol represents the mean from two to three assays. The dashed lines represent a −0.5 log₁₀ change, the threshold considered positive in the assay (see Table 2). Against strain NZ98/254, 11% of the preimmunization sera were positive for PP, and this proportion increased to 61% after immunization (P < 0.001). For strain S3032, the proportions of sera with PP activity before and after immunization were 42% and 94%, respectively (P < 0.001).

FIG. 4.

Serum PP activity in relation to complement-mediated serum bactericidal titers. Each point represents the mean ± standard deviation for three assays performed on different days on 1:4 dilutions of postimmunization sera from individual subjects. (A) Strain NZ98/254; (B) strain S3032. Positive PP (dashed lines) was defined by a ≥0.5 log₁₀ decrease in CFU/ml at 90 min from the CFU/ml of the negative control at time zero. The log₁₀ changes in CFU/ml in the whole-blood assay for many of the sera with SBA titers of <1:4 and positive PP (center panels) were similar in magnitude to those observed for sera with bactericidal titers of ≥1:4 and positive PP (upper panels). Note that there was one subject with a postimmunization bactericidal titer of 1:7 and negative PP (−0.4 log₁₀ decrease) against strain NZ98/254. Since no other sera exhibited positive SBA and negative PP, a fourth panel with the single data point is not shown.

FIG. 5.

Summary of the proportions of immunized subjects with titers of ≥1:4 in SBA, OPA, or PP assays. The bars represent the proportions of preimmunization (open bars) or postimmunization (filled bars) sera that were positive when tested at a 1:4 dilution in each assay. Error bars, 95% confidence intervals (CI). A positive serum SBA or OPA titer was defined as a 50% decrease, after a 60-min incubation of bacteria, from the CFU/ml at time zero. Positive PP activity was defined as a ≥0.5 log₁₀ decrease, after a 90-min incubation, from the CFU/ml at time zero. Depending on the strain, postimmunization PP activity was 2.6- to 2.8-fold more frequent then an SBA titer of ≥1:4.