This is the first study in which multiple immunoassays have been compared to evaluate the serum antibody responses induced by either a group A conjugate vaccine or a bivalent meningococcal A/C polysaccharide vaccine.
Two serologic assays, the SBA assay and the group-specific ELISA, have standardized protocols, and the results from multicenter collaborative studies have shown that different laboratories can obtain comparable results (3
The SBA assay measures the ability of serum antibody to kill meningococci in the presence of complement. Many factors can influence the results of measurement of meningococcal bactericidal activity; these include the choice of the bacterial test strain, conditions for growth of the bacteria, and the source of the exogenous complement. Classic studies in the 1960s demonstrated a correlation between SBA activity and protection against meningococcal disease; however, these studies used normal human serum, which lacked intrinsic bactericidal activity, as the complement source (7
). Following international workshops on the development of standardized laboratory protocols, subsequent multicenter collaborative studies used baby rabbit complement (21
) that is commercially available (Pel-Freez). Until now, no meningococcal serogroup A hSBA interlaboratory studies have been conducted. In this study, significantly different antibody titers were observed when SBA titers were generated using baby rabbit serum than when SBA titers were generated using human serum as the complement source, as has been noted in previous studies (21
). The modest correlation between hSBA and rSBA observed in this study agrees with the correlation noted by Norheim et al. (22
). Interestingly, a significant difference between the two vaccine groups was found at 4 weeks postvaccination by the hSBA assay and by both SBA assays at 48 weeks. Despite the difference in the magnitude of the SBA titers obtained with human and rabbit complement, a high proportion of responders were observed in the PsA-TT group by either assay when these data were analyzed by titers of ≥8 and by a ≥4-fold titer rise. Goldschneider et al. (7
) demonstrated an hSBA titer of ≥4 to correlate with protection from meningococcal disease, but due to the limit of detection of the hSBA assay used in this study being a titer of 4, a more conservative level of ≥8 was used to analyze the hSBA data in order not to overestimate protection. Despite standardization of MenA rSBA assays (21
), serologic correlates of protection have not been established for MenA; however, by extrapolation from protective MenC rSBA titers (2
), SBA titers of ≥8 have been used to indicate protection. With the use of a threshold titer of ≥8 for both SBA assays, at 4 weeks postvaccination all subjects in both groups had rSBA titers of ≥8 while 92% and 74% of subjects in the PsA-TT and PsA/C vaccine groups, respectively, had hSBA titers of ≥8. With the use of a higher discriminatory titer of ≥128 for rSBA, at 4 weeks postvaccination all subjects in both groups had rSBA titers above this level. The proportion of subjects demonstrating a ≥4-fold rise in rSBA titers at 4 weeks postvaccination was slightly higher in the PsA-TT vaccine group than in the PsA/C vaccine group (83% versus 72%, respectively), as were these proportions for hSBA titers, which were 87% and 48%, respectively. Among subjects demonstrating a ≥4-fold rise in titer from baseline to 4 weeks postvaccination, there was moderate agreement between rSBA and hSBA (>65%).
Antibody-dependent complement-mediated bactericidal killing is considered a surrogate of protection in meningococcal disease. However, the only correlate of protection for group A is that which was derived from the Finnish efficacy trials of a meningococcal group A polysaccharide vaccine (16
). For this correlation, a group A-specific Ig concentration of 2 μg/ml, as determined by radioimmunoassay, was derived from the mean level in unimmunized adults (18
) who were assumed to be protected, and this level corresponded to that achieved by the majority of individuals in the age groups in which the vaccine was shown to be effective (23
). In our study, group A-specific IgG was measured by two methods, the standardized ELISA (4
) and a multiplex bead assay (14
). A good correlation was observed between the group A-specific IgG concentrations obtained by these two methods, as was previously reported by Lal et al. (14
), although the IgG results from the multiplex bead assay were consistently lower. This is probably due to differences in reaction kinetics, with the ELISA detecting both high- and low-avidity antibodies and the multiplex bead assay detecting high-avidity antibody and hence overall a lower MenA-specific IgG concentration.
A moderate correlation between rSBA and group A-specific IgG was observed in this study, which agrees with correlations previously described (17
). Results of an earlier study that showed improved correlation when a modified ELISA that selected for high-avidity antibodies was used (12
) suggest that this moderate correlation between rSBA and group A-specific IgG is due to the extent to which antibody binding assays measure both functional and nonfunctional IgG, with the latter usually being of low avidity. Bactericidal activity is determined not only by the quantity but also by the quality of antibody, e.g., the isotype, subclass, and avidity. Despite various correlation coefficients between the SBA and group A-specific IgG, similar proportions of subjects are regarded as protected (hSBA titer of ≥8, rSBA titer of ≥8, and MenA-specific IgG titer of ≥2 μg/ml) by the different assays. Four weeks postvaccination, there was an agreement between 92% of subjects being regarded as protected by the four different assays for the PsA-TT group whereas there was only an agreement between the four assays for 68% of subjects in the PsA/C group. Norheim et al. (22
) observed a correlation between hSBA titers and group A-specific IgG concentrations in Ethiopian case sera similar to that which was observed in this study for the PsA/C group. Interestingly, the Pearson correlation coefficient between the hSBA and group A-specific IgG (either method) was higher for the PsA-TT group than for the PsA/C group, indicating the higher quality of antibody induced by the PsA-TT vaccine.
Demonstration of killing of meningococci by opsonophagocytosis (24
), coupled with the establishment of the OPA as the surrogate of protection for Streptococcus pneumoniae
), has led to the development of an OPA against meningococci (1
). Immunization with meningococcal polysaccharide vaccines elicits complement-dependent serum bactericidal and opsonizing antibodies, with both mechanisms operating simultaneously to clear meningococci (7
). In this study there was little correlation between the two OPA assays, possibly due to methodologic differences. The CDC OPA assay measures internalization and uses antigen-coated particles and rabbit complement while the NIPH OPA assay measures respiratory burst and employs live meningococci and human complement. In addition, different effector cells are used in these OPA assays. Poor correlations were observed between either the CDC or the NIPH OPA assay and all other assays, although the reasons for this discrepancy are unclear. This is in contrast to measurement of levels of OPA against group B meningococci, which usually demonstrate a good correlation with SBA and IgG levels (1
). Also, OPA levels were found to rapidly decline at 1 year following vaccination, which was not the case with the rSBA. The decline at week 48 to levels below prevaccination levels in the CDC OPA assay is puzzling, as this was not observed for any other assays and the week 24 and 48 samples were tested at the same time, so that it appears that the unexpected inexplicability of the result is not likely to be due to the testing method. These data call into question the relevance of the OPA assays as they currently exist in measuring the immune response to meningococci.
In summary, the best linear relationship was observed between group A-specific IgG levels as determined by ELISA and by the multiplex bead assay. Only a moderate correlation was observed between the hSBA and rSBA; however, the assays were similar regarding the proportions of subjects that demonstrated a fourfold rise in titer from baseline to 4 weeks postvaccination. Only a weak correlation was found between the two OPAs examined. Both OPAs were shown to correlate weakly with each other and showed little or no correlation with SBA or IgG concentration. Other than the two antibody binding assays (ELISA and multiplex bead assay), none of the serogroup A immunoassays showed sufficiently high correlation to consider one assay able to predict or represent another assay. Without an accompanying study of vaccine efficacy, the question of how each assay relates to protection cannot be definitively determined. However, this study may be useful in examining postimmunization efficacy data in relation to serogroup A immunoassays. We recommend that for future group A vaccine trials, a rational approach is to determine functional bactericidal activity as a primary endpoint, while IgG can provide additional supporting evidence of immunogenicity. This study suggests that when rSBA is used as a primary serologic assay for all sera, hSBA performed on a subset provides additional supportive data and additional information may be provided by measuring IgG-specific antibody. World Health Organization requirements have been established for meningococcal polysaccharide vaccines using rabbit complement, but currently no criteria have been set for data generated using human complement (27
). The difference in titers obtained when rabbit complement is used and when human complement is used in the SBA assay may be investigated further by the addition of human factor H to rabbit complement.